Neprilysin inhibitors

ABSTRACT

In one aspect, the invention relates to compounds having the formula: 
                         
where R 1 -R 6 , a, b, and Z are as defined in the specification, or a pharmaceutically acceptable salt thereof. These compounds have neprilysin inhibition activity. In another aspect, the invention relates to pharmaceutical compositions comprising such compounds; methods of using such compounds; and processes and intermediates for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/180,479, filed onFeb. 14, 2014, now U.S. Pat. No. 9,150,500, which a continuation of U.S.Ser. No. 13/666,538, filed on Nov. 1, 2012, now U.S. Pat. No. 8,691,868,which claims the benefit of U.S. Provisional Application No. 61/554,625,filed on Nov. 2, 2011; the entire disclosures of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to novel compounds havingneprilysin-inhibition activity. The invention also relates topharmaceutical compositions comprising such compounds, processes andintermediates for preparing such compounds and methods of using suchcompounds to treat diseases such as hypertension, heart failure,pulmonary hypertension, and renal disease.

State of the Art

Neprilysin (neutral endopeptidase, EC 3.4.24.11) (NEP), is anendothelial membrane bound Zn²⁺ metallopeptidase found in many organsand tissues, including the brain, kidneys, lungs, gastrointestinaltract, heart, and the peripheral vasculature. NEP degrades andinactivates a number of endogenous peptides, such as enkephalins,circulating bradykinin, angiotensin peptides, and natriuretic peptides,the latter of which have several effects including, for example,vasodilation and natriuresis/diuresis, as well as inhibition of cardiachypertrophy and ventricular fibrosis. Thus, NEP plays an important rolein blood pressure homeostasis and cardiovascular health.

NEP inhibitors, such as thiorphan, candoxatril, and candoxatrilat, havebeen studied as potential therapeutics. Compounds that inhibit both NEPand angiotensin-I converting enzyme (ACE) are also known, and includeomapatrilat, gempatrilat, and sampatrilat. Referred to as vasopeptidaseinhibitors, this latter class of compounds is described in Robl et al.(1999) Exp. Opin. Ther. Patents 9(12): 1665-1677.

Ksander et al. (1995) J. Med. Chem. 38:1689-1700 describes dicarboxylicacid dipeptide NEP inhibitors of the formula:

Compound R IC₅₀ (nM) 21g —C(O)—CH₂—COOH 92 21a (R,S) —C(O)—(CH₂)₂—COOH 521b (S,R) —C(O)—(CH₂)₂—COOH 190 21c (R,R) —C(O)—(CH₂)₂—COOH 700 21d(S,S) —C(O)—(CH₂)₂—COOH 27 21e —C(O)—(CH₂)₃—COOH 90 21f—C(O)—(CH₂)₄—COOH 324Compound 21a, which has a succinic acid substituent, is the most activecompound, with an IC₅₀ of 5 nM. The authors observed that “the succinincacid in the P₂′ site appears to be optimal since extension of thecarboxylic acid chain by one (21e) and two (21f) methylene unitsdecreased activity 18- and 65-fold.” The authors further noted that“decreasing the chain length by one methylene (21g) also showed an18-fold decrease in activity.” (page 1692, 2^(nd) column)

SUMMARY OF THE INVENTION

The present invention provides novel compounds that have been found topossess neprilysin (NEP) enzyme inhibition activity. Accordingly,compounds of the invention are expected to be useful and advantageous astherapeutic agents for treating conditions such as hypertension andheart failure.

One aspect of the invention relates to a compound of formula I:

where:

R¹ is selected from H, —C₁₋₈alkyl, —C₁₋₃allcylene-C₆₋₁₀aryl,—C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃,—C₁₋₆alkylene-OC(O)R¹⁰, —C₁₋₆alkylene-NR¹¹R¹², —C₁₋₆alkylene-C(O)R¹³,—C₀₋₆alkylenemorpholinyl, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R¹⁰ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹¹R¹², —CH(R¹⁵)—NH₂,—CH(R¹⁵)—NHC(O)O—C₁₋₆alkyl, and —CH(NH₂)CH₂COOCH₃; and R¹¹ and R¹² areindependently selected from H, —C₁₋₆alkyl, and benzyl; or R¹¹ and R¹²are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—; R¹³is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹¹R¹²; and R¹⁴ is—C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; R¹⁵ is H, —CH₃, —CH(CH₃)₂,phenyl, or benzyl;

R² is —OR²¹ or —CH₂OR²¹; and R³ is H or —CH₃; where R²¹ is H,—C(O)—C₁₋₆alkyl, —C(O)—CH(R²²)—NH₂, —C(O)—CH(R²²)—NHC(O)O—C₁₋₆alkyl, or—P(O)(OR²³)₂; R²² is H, —CH₃, —CH(CH₃)₂, phenyl, or benzyl; R²³ is H,—C₁₋₆alkyl, or phenyl; or

R² is taken together with R¹ to form —OCR¹⁵R¹⁶— or —CH₂O—CR¹⁵R¹⁶—, andR³ is selected from H and —CH₃, where R¹⁵ and R¹⁶ are independentlyselected from H, —C₁₋₆alkyl, and —O—C₃₋₇cycloalkyl, or R¹⁵ and R¹⁶ aretaken together to form ═O; or

R² is taken together with R³ to form —CH₂—O—CH₂— or —CH₂—CH₂—; or

R² and R³ are both —CH₃;

Z is selected from —CH— and —N—;

R⁴ is selected from H, —C₁₋₈alkyl, —C₁₋₃alkylene-O—C₁₋₈alkyl,—C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-O—C₆₋₁₀aryl,—C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃,—C₁₋₆alkylene-OC(O)R⁴⁰, —C₁₋₆alkylene-NR⁴¹R⁴², —C₁₋₆alkylene-C(O)R⁴³,—C₀₋₆alkylenemorpholinyl, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R⁴⁰ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR⁴¹R⁴², —CH(R⁴⁵)—NH₂,—CH(R⁴⁵)—NHC(O)O—C₁₋₆alkyl, and —CH(NH₂)CH₂COOCH₃; and R⁴¹ and R⁴² areindependently selected from H, —C₁₋₆alkyl, and benzyl; or R⁴¹ and R⁴²are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—; R⁴³is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR⁴¹R⁴²; and R⁴⁴ is—C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; R⁴⁵ is H, —CH₃, —CH(CH₃)₂,phenyl, or benzyl;

a is 0 or 1; R⁵ is selected from halo, CH₃, CF₃, and —CN;

b is 0 or an integer from 1 to 3; each R⁶ is independently selected fromhalo, —OH, —CH₃, OCH₃, —CN, and —CF₃;

where each alkyl group in R¹ and R⁴ is optionally substituted with 1 to8 fluoro atoms; and

where the methylene linker on the biphenyl is optionally substitutedwith one or two —C₁₋₆alkyl groups or cyclopropyl;

or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention. Such compositions may optionally contain other therapeuticagents. Accordingly, in yet another aspect of the invention, apharmaceutical composition comprises a compound of the invention as thefirst therapeutic agent, one or more secondary therapeutic agent, and apharmaceutically acceptable carrier. Another aspect of the inventionrelates to a combination of active agents, comprising a compound of theinvention and a second therapeutic agent. The compound of the inventioncan be formulated together or separately from the additional agent(s).When formulated separately, a pharmaceutically acceptable carrier may beincluded with the additional agent(s). Thus, yet another aspect of theinvention relates to a combination of pharmaceutical compositions, thecombination comprising: a first pharmaceutical composition comprising acompound of the invention and a first pharmaceutically acceptablecarrier; and a second pharmaceutical composition comprising a secondtherapeutic agent and a second pharmaceutically acceptable carrier. Inanother aspect, the invention relates to a kit containing suchpharmaceutical compositions, for example where the first and secondpharmaceutical compositions are separate pharmaceutical compositions.

Compounds of the invention possess NEP enzyme inhibition activity, andare therefore expected to be useful as therapeutic agents for treatingpatients suffering from a disease or disorder that is treated byinhibiting the NEP enzyme or by increasing the levels of its peptidesubstrates. Thus, one aspect of the invention relates to a method oftreating patients suffering from a disease or disorder that is treatedby inhibiting the NEP enzyme, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Anotheraspect of the invention relates to a method of treating hypertension,heart failure, or renal disease, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Stillanother aspect of the invention relates to a method for inhibiting a NEPenzyme in a mammal comprising administering to the mammal, a NEPenzyme-inhibiting amount of a compound of the invention.

Since compounds of the invention possess NEP inhibition activity, theyare also useful as research tools. Accordingly, one aspect of theinvention relates to a method of using a compound of the invention as aresearch tool, the method comprising conducting a biological assay usinga compound of the invention. Compounds of the invention can also be usedto evaluate new chemical compounds. Thus another aspect of the inventionrelates to a method of evaluating a test compound in a biological assay,comprising: (a) conducting a biological assay with a test compound toprovide a first assay value; (b) conducting the biological assay with acompound of the invention to provide a second assay value; wherein step(a) is conducted either before, after or concurrently with step (b); and(c) comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include a NEP enzymeinhibition assay. Still another aspect of the invention relates to amethod of studying a biological system or sample comprising a NEPenzyme, the method comprising: (a) contacting the biological system orsample with a compound of the invention; and (b) determining the effectscaused by the compound on the biological system or sample.

Yet another aspect of the invention relates to processes andintermediates useful for preparing compounds of the invention.Accordingly, another aspect of the invention relates to a process ofpreparing compounds of formula I, comprising the step of coupling acompound of formula 1 with a compound of formula 2:

to produce a compound of formula I; where P is H or an amino-protectinggroup selected from t-butoxycarbonyl, trityl, benzyloxycarbonyl,9-fluorenylmethoxycarbonyl, formyl, trimethylsilyl, andt-butyldimethylsilyl; and where the process further comprisesdeprotecting the compound of formula 1 when P is an amino protectinggroup; and where R¹-R⁶, a, b, and Z are as defined for formula I.Another aspect of the invention relates to a process of preparing apharmaceutically acceptable salt of a compound of formula I, comprisingcontacting a compound of formula I in free acid or base form with apharmaceutically acceptable base or acid. In other aspects, theinvention relates to products prepared by any of the processes describedherein, as well as novel intermediates used in such process. In oneaspect of the invention novel intermediates have formula 1 or a saltthereof, as defined herein.

Yet another aspect of the invention relates to the use of a compound offormula I or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament, especially for the manufacture of amedicament useful for treating hypertension, heart failure, or renaldisease. Another aspect of the invention relates to use of a compound ofthe invention for inhibiting a NEP enzyme in a mammal Still anotheraspect of the invention relates to the use of a compound of theinvention as a research tool. Other aspects and embodiments of theinvention are disclosed herein.

A particular group of compounds of formula I are those disclosed in U.S.Provisional Application No. 61/554,625, filed on Nov. 2, 2011. Thisgroup includes compounds of formula I; wherein:

where: R¹ is selected from H, —C₁₋₈alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl,—C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃,—C₁₋₆alkylene-OC(O)R¹⁰, —C₁₋₆alkylene-NR¹¹R¹², —C₁₋₆alkylene-C(O)R¹³,—C₀₋₆alkylenemorpholinyl, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R¹⁰ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹¹R¹², —CH[CH(CH₃)₂]—NH₂,—CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl, and —CH(NH₂)CH₂COOCH₃; and R¹¹ and R¹²are independently selected from H, —C₁₋₆alkyl, and benzyl; or R¹¹ andR¹² are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—;R¹³ is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹¹R¹²; and R¹⁴ is—C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; R² is selected from —OH, —CH₂OH,—OP(O)(OH)₂, and —CH₂OP(O)(OH)₂; and R³ is selected from H and —CH₃; orR² is taken together with R¹ to form —OCR¹⁵R¹⁶— or —CH₂O—CR¹⁵R¹⁶—, andR³ is selected from H and —CH₃, where R¹⁵ and R¹⁶ are independentlyselected from H, —C₁₋₆alkyl, and —O—C₃₋₇cycloalkyl, or R¹⁵ and R¹⁶ aretaken together to form ═O; or R² is taken together with R³ to form—CH₂—O—CH₂— or —CH₂—CH₂—; or R² and R³ are both —CH₃; Z is selected from—CH— and —N—; R⁴ is selected from H, —C₁₋₈alkyl,—C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl,—[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R⁴⁶, —C₁₋₆alkylene-NR⁴¹R⁴²,—C₁₋₆alkylene-C(O)R⁴³, —C₀₋₆alkylenemorpholinyl,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R⁴⁰ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR⁴¹R⁴², —CH[CH(CH₃)₂]—NH₂,—CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl, and —CH(NH₂)CH₂COOCH₃; and R⁴¹ and R⁴²are independently selected from H, —C₁₋₆alkyl, and benzyl; or R⁴¹ andR⁴² are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—;R⁴³ is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR⁴¹R⁴²; and R⁴⁴ is—C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; a is 0 or 1; R⁵ is selected fromhalo, —CH₃, —CF₃, and —CN; b is 0 or an integer from 1 to 3; each R⁶ isindependently selected from halo, —OH, —CH₃, —OCH₃, and —CF₃; and whereeach alkyl group in R¹ and R⁴ is optionally substituted with 1 to 8fluoro atoms; and; where the methylene linker on the biphenyl isoptionally substituted with one or two —C₁₋₆alkyl groups or cyclopropyl;or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE INVENTION Definitions

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an,” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms and include, for example,—C₁₋₄alkyl, —C₁₋₅alkyl, —C₂₋₅alkyl, —C₁₋₆alkyl, —C₁₋₈alkyl, and—C₁₋₁₀alkyl. Representative alkyl groups include, by way of example,methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl,n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown preceding the term assubscript. For example, the term “—C₁₋₆alkyl” means an alkyl grouphaving from 1 to 6 carbon atoms, and the term “—C₃₋₇cycloalkyl” means acycloalkyl group having from 3 to 7 carbon atoms, respectively, wherethe carbon atoms are in any acceptable configuration.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 0 to 10 carbon atoms and include, forexample, —C₀₋₁alkylene-, —C₀₋₆alkylene-, —C₁₋₃allylene-, and—C₁₋₆alkylene-. Representative alkylene groups include, by way ofexample, methylene, ethane-1,2-diyl (“ethylene”), propane-1,2-diyl,propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl and the like. It isunderstood that when the alkylene term include zero carbons such as—C₀₋₁alkylene-, such terms are intended to include the absence of carbonatoms, that is, the alkylene group is not present except for a covalentbond attaching the groups separated by the alkylene term.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or one or more fused rings. Fused ring systemsinclude those that are fully unsaturated (e.g., naphthalene) as well asthose that are partially unsaturated (e.g.,1,2,3,4-tetrahydronaphthalene). Unless otherwise defined, such arylgroups typically contain from 6 to 10 carbon ring atoms and include, forexample, —C₆₋₁₀aryl. Representative aryl groups include, by way ofexample, phenyl and naphthalene-1-yl, naphthalene-2-yl, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms and include, for example,—C₃₋₅cycloalkyl, —C₃₋₆cycloalkyl and —C₃₋₇cycloalkyl. Representativecycloalkyl groups include, by way of example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heteroaryl” is intended to mean a monovalent unsaturated(aromatic) heterocycle having a single ring or two fused rings.Monovalent unsaturated heterocycles are also commonly referred to as“heteroaryl” groups. Unless otherwise defined, heteroaryl groupstypically contain from 5 to 10 total ring atoms, of which 1 to 9 arering carbon atoms, and 1 to 4 are ring heteroatoms, and include, forexample, —C₁₋₉heteroaryl and —C₅₋₉heteroaryl. Representative heteroarylgroups include, by way of example, pyrrole (e.g., 3-pyrrolyl and2H-pyrrol-3-yl), imidazole (e.g., 2-imidazolyl), furan (e.g., 2-furyland 3-furyl), thiophene (e.g., 2-thienyl), triazole (e.g.,1,2,3-triazolyl and 1,2,4-triazolyl), pyrazole (e.g., 1H-pyrazol-3-yl),oxazole (e.g., 2-oxazolyl), isoxazole (e.g., 3-isoxazolyl), thiazole(e.g., 2-thiazolyl and 4-thiazolyl), and isothiazole (e.g.,3-isothiazolyl), pyridine (e.g., 2-pyridyl, 3-pyridyl, and 4-pyridyl),pyridylimidazole, pyridyltriazole, pyrazine, pyridazine (e.g.,3-pyridazinyl), pyrimidine (e.g., 2-pyrimidinyl), tetrazole, triazine(e.g., 1,3,5-triazinyl), indolyle (e.g., 1H-indol-2-yl, 1H-indol-4-yland 1H-indol-5-yl), benzofuran (e.g., benzofuran-5-yl), benzothiophene(e.g., benzo[b]thien-2-yl and benzo[b]thien-5-yl), benzimidazole,benzoxazole, benzothiazole, benzotriazole, quinoline (e.g., 2-quinolyl),isoquinoline, quinazoline, quinoxaline and the like.

The term “optionally substituted” means that group in question may beunsubstituted or it may be substituted one or several times, such as 1to 3 times, or 1 to 5 times, or 1 to 8 times. For example, an alkylgroup that is “optionally substituted” with fluoro atoms may beunsubstituted, or it may contain 1, 2, 3, 4, 5, 6, 7, or 8 fluoro atoms.Similarly, a group that is “optionally substituted” with one or two—C₁₋₆alkyl groups, may be unsubstituted, or it may contain one or two—C₁₋₆alkyl groups.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used. For example, if onestructure is depicted, it is understood that all stereoisomer andtautomer forms are encompassed, unless stated otherwise.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug administration.

The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (for example, salts having acceptable mammalian safetyfor a given dosage regime). However, it is understood that the saltscovered by the invention are not required to be pharmaceuticallyacceptable salts, such as salts of intermediate compounds that are notintended for administration to a patient. Pharmaceutically acceptablesalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. In addition, when a compound of formula I contains both a basicmoiety, such as an amine, pyridine or imidazole, and an acidic moietysuch as a carboxylic acid or tetrazole, zwitterions may be formed andare included within the term “salt” as used herein. Salts derived frompharmaceutically acceptable inorganic bases include ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic, manganous,potassium, sodium, and zinc salts, and the like. Salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (for example, citric, gluconic, glycolic,lactic, lactobionic, malic, and tartaric acids), aliphaticmonocarboxylic acids (for example, acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (for example, aspartic andglutamic acids), aromatic carboxylic acids (for example, benzoic,p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylaceticacids), aromatic hydroxyl acids (for example, o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids(for example, fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(for example, benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like.

As used herein, the term “prodrug” is generally intended to mean aninactive precursor of a drug that is converted into its active form inthe body under physiological conditions, for example, by normalmetabolic processes. Such compounds may not possess pharmacologicalactivity at NEP, but may be administered orally or parenterally andthereafter metabolized in the body to form compounds that arepharmacologically active at NEP. When orally administered, suchcompounds may also provide a better fraction absorbed (i.e., better pKproperties) for renal delivery, as compared to oral administration ofthe active form. Exemplary prodrugs include esters such asC₁₋₆alkylesters and aryl-C₁₋₆alkylesters. In one embodiment, the activecompound has a free carboxyl and the prodrug is an ester derivativethereof, i.e., the prodrug is an ester such as —C(O)OCH₂CH₃. Such esterprodrugs are then converted by solvolysis or under physiologicalconditions to be the free carboxyl compound. The term “prodrug” is alsointended to include a less active precursor of a drug that is convertedinto a more active form in the body. For example, certain prodrugs maypossess pharmacological activity at NEP, but not necessarily at thedesired level; such compounds are converted in the body into a formhaving the desired level of activity. The term is also intended toinclude certain protected derivatives of compounds of formula I that maybe made prior to a final deprotection stage. Thus, all protectedderivatives and prodrugs of compounds formula I are included within thescope of the invention.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof, thatis, the amount of drug needed to obtain the desired therapeutic effect.For example, a therapeutically effective amount for treatinghypertension is an amount of compound needed to, for example, reduce,suppress, eliminate, or prevent the symptoms of hypertension, or totreat the underlying cause of hypertension. In one embodiment, atherapeutically effective amount is that amount of drug needed to reduceblood pressure or the amount of drug needed to maintain normal bloodpressure. On the other hand, the term “effective amount” means an amountsufficient to obtain a desired result, which may not necessarily be atherapeutic result. For example, when studying a system comprising a NEPenzyme, an “effective amount” may be the amount needed to inhibit theenzyme.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as hypertension) in apatient, such as a mammal (particularly a human) that includes one ormore of the following: (a) preventing the disease or medical conditionfrom occurring, i.e., preventing the reoccurrence of the disease ormedical condition or prophylactic treatment of a patient that ispre-disposed to the disease or medical condition; (b) ameliorating thedisease or medical condition, i.e., eliminating or causing regression ofthe disease or medical condition in a patient; (c) suppressing thedisease or medical condition, i.e., slowing or arresting the developmentof the disease or medical condition in a patient; or (d) alleviating thesymptoms of the disease or medical condition in a patient. For example,the term “treating hypertension” would include preventing hypertensionfrom occurring, ameliorating hypertension, suppressing hypertension, andalleviating the symptoms of hypertension (for example, lowering bloodpressure). The term “patient” is intended to include those mammals, suchas humans, that are in need of treatment or disease prevention or thatare presently being treated for disease prevention or treatment of aspecific disease or medical condition, as well as test subjects in whichcompounds of the invention are being evaluated or being used in anassay, for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

In one aspect, the invention relates to compounds of formula I:

or a pharmaceutically acceptable salt thereof.

As used herein, the term “compound of the invention” includes allcompounds encompassed by formula I such as the species embodied informulas Ia and Ib, as well as the compounds encompassed by formulasIIa-IIk, IIIa-IIIb, and IVa-IVd. In addition, the compounds of theinvention may also contain several basic or acidic groups (for example,amino or carboxyl groups) and therefore, such compounds can exist as afree base, free acid, or in various salt forms. All such salt forms areincluded within the scope of the invention. Furthermore, the compoundsof the invention may also exist as prodrugs. Accordingly, those skilledin the art will recognize that reference to a compound herein, forexample, reference to a “compound of the invention” or a “compound offormula I” includes a compound of formula I as well as pharmaceuticallyacceptable salts and prodrugs of that compound unless otherwiseindicated. Further, the term “or a pharmaceutically acceptable saltand/or prodrug thereof” is intended to include all permutations of saltsand prodrugs, such as a pharmaceutically acceptable salt of a prodrug.Furthermore, solvates of compounds of formula I are included within thescope of this invention.

The compounds of formula I may contain one or more chiral centers andtherefore, these compounds may be prepared and used in variousstereoisomeric forms. Accordingly, the invention also relates to racemicmixtures, pure stereoisomers (e.g., enantiomers and diastereoisomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted herein without anystereochemistry, it is understood that all possible stereoisomers areencompassed by such structure. Thus, for example, the terms “compound offormula I,” “compounds of formula II,” and so forth, are intended toinclude all possible stereoisomers of the compound. Similarly, when aparticular stereoisomer is shown or named herein, it will be understoodby those skilled in the art that minor amounts of other stereoisomersmay be present in the compositions of the invention unless otherwiseindicated, provided that the utility of the composition as a whole isnot eliminated by the presence of such other isomers. Individualstereoisomers may be obtained by numerous methods that are well known inthe art, including chiral chromatography using a suitable chiralstationary phase or support, or by chemically converting them intodiastereoisomers, separating the diastereoisomers by conventional meanssuch as chromatography or recrystallization, then regenerating theoriginal stereoisomer.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds ofthe invention are included within the scope of the invention unlessotherwise specified.

More specifically, compounds of formula I can contain at least twochiral centers when the “Z” moiety is —CH—, and can contain at least onechiral center when the “Z” moiety is —N—. These chiral centers areindicated by the symbols * and ** in the following formulas Ia and Ib:

Note however, that there is no * chiral center when R² is taken togetherwith R³ to form —CH₂—O—CH₂— or —CH₂—CH₂—, or R² and R³ are both —CH₃.

In one stereoisomer of the compound of formula Ia, both carbon atomsidentified by the * and ** symbols have the (R) configuration. In thisembodiment, compounds have the (R,R) configuration at the * and **carbon atoms or are enriched in a stereoisomeric form having the (R,R)configuration at these carbon atoms. In another stereoisomer of thecompound of formula Ia, both carbon atoms identified by the * and **symbols have the (S) configuration. In this embodiment, compounds havethe (S,S) configuration at the * and ** carbon atoms or are enriched ina stereoisomeric form having the (S,S) configuration at these carbonatoms. In yet another stereoisomer of the compound of formula Ia, thecarbon atom identified by the symbol * has the (S) configuration and thecarbon atom identified by the symbol ** has the (R) configuration. Inthis embodiment, compounds have the (S,R) configuration at the * and **carbon atoms or are enriched in a stereoisomeric form having the (S,R)configuration at these carbon atoms. In still another stereoisomer ofthe compound of formula Ia, the carbon atom identified by the symbol *has the (R) configuration and the carbon atom identified by the symbol** has the (S) configuration. In this embodiment, compounds have the(R,S) configuration at the * and ** carbon atoms or are enriched in astereoisomeric form having the (R,S) configuration at these carbonatoms.

In one stereoisomer of the compound of formula Ib, the carbon atomidentified by the * symbol has the (R) configuration. In thisembodiment, compounds have the (R) configuration at the * carbon atom orare enriched in a stereoisomeric form having the (R) configuration atthis carbon atom. In another stereoisomer of the compound of formula Ib,the carbon atom identified by the * symbol has the (S) configuration. Inthis embodiment, compounds have the (S) configuration at the * carbonatom or are enriched in a stereoisomeric form having the (S)configuration at this carbon atom.

Formula R² R³ * ** Ia-1 —OR²¹ H (R) (R) Ia-1 —OR²¹ —CH₃ (R) (R) Ia-1—CH₂OR²¹ H (S) (S) Ia-1 —CH₂OR²¹ —CH₃ (S) (R) Ia-1 R² is taken togetherwith R¹ to form H (R) (R) —OCR¹⁵R¹⁶— Ia-1 R² is taken together with R¹to form —CH₃ (R) (R) —OCR¹⁵R¹⁶— Ia-1 R² is taken together with R¹ toform H (S) (S) —CH₂O—CR¹⁵R¹⁶— Ia-1 R² is taken together with R¹ to form—CH₃ (R) (S) —CH₂O—CR¹⁵R¹⁶— Ia-2 —OR²¹ H (S) (S) Ia-2 —OR²¹ —CH₃ (S) (S)Ia-2 —CH₂OR²¹ H (R) (R) Ia-2 —CH₂OR²¹ —CH₃ (R) (S) Ia-2 R² is takentogether with R¹ to form H (S) (S) —OCR¹⁵R¹⁶— Ia-2 R² is taken togetherwith R¹ to form —CH₃ (S) (S) —OCR¹⁵R¹⁶— Ia-2 R² is taken together withR¹ to form H (R) (R) —CH₂O—CR¹⁵R¹⁶— Ia-2 R² is taken together with R¹ toform —CH₃ (S) (R) —CH₂O—CR¹⁵R¹⁶— Ia-3 —OR²¹ H (S) (R) Ia-3 —OR²¹ —CH₃(S) (R) Ia-3 —CH₂OR²¹ H (R) (S) Ia-3 —CH₂OR²¹ —CH₃ (R) (R) Ia-3 R² istaken together with R¹ to form H (R) (S) —OCR¹⁵R¹⁶— Ia-3 R² is takentogether with R¹ to form —CH₃ (R) (S) —OCR¹⁵R¹⁶— Ia-3 R² is takentogether with R¹ to form H (S) (R) —CH₂O—CR¹⁵R¹⁶— Ia-3 R² is takentogether with R¹ to form —CH₃ (R) (R) —CH₂O—CR¹⁵R¹⁶— Ia-4 —OR²¹ H (R)(S) Ia-4 —OR²¹ —CH₃ (R) (S) Ia-4 —CH₂OR²¹ H (S) (R) Ia-4 —CH₂OR²¹ —CH₃(S) (S) Ia-4 R² is taken together with R¹ to form H (S) (R) —OCR¹⁵R¹⁶—Ia-4 R² is taken together with R¹ to form —CH₃ (S) (R) —OCR¹⁵R¹⁶— Ia-4R² is taken together with R¹ to form H (R) (S) —CH₂O—CR¹⁵R¹⁶— Ia-4 R² istaken together with R¹ to form —CH₃ (S) (S) —CH₂O—CR¹⁵R¹⁶— Ib-1 —OR²¹ H(R) NA Ib-1 —OR²¹ —CH₃ (R) NA Ib-1 —CH₂OR²¹ H (S) NA Ib-1 —CH₂OR²¹ —CH₃(S) NA Ib-1 R² is taken together with R¹ to form H (R) NA —OCR¹⁵R¹⁶—Ib-1 R² is taken together with R¹ to form —CH₃ (R) NA —OCR¹⁵R¹⁶— Ib-1 R²is taken together with R¹ to form H (S) NA —CH₂O—CR¹⁵R¹⁶— Ib-1 R² istaken together with R¹ to form —CH₃ (S) NA —CH₂O—CR¹⁵R¹⁶— Ib-2 —OR²¹ H(S) NA Ib-2 —OR²¹ —CH₃ (S) NA Ib-2 —CH₂OR²¹ H (R) NA Ib-2 —CH₂OR²¹ —CH₃(R) NA Ib-2 R² is taken together with R¹ to form H (S) NA —OCR¹⁵R¹⁶—Ib-2 R² is taken together with R¹ to form —CH₃ (S) NA —OCR¹⁵R¹⁶— Ib-2 R²is taken together with R¹ to form H (R) NA —CH₂O—CR¹⁵R¹⁶— Ib-2 R² istaken together with R¹ to form —CH₃ (R) NA —CH₂O—CR¹⁵R¹⁶—

In some embodiments, in order to optimize the therapeutic activity ofthe compounds of the invention, e.g., to treat hypertension, it may bedesirable that the carbon atoms identified by the * and ** symbols havea particular configuration or are enriched in a stereoisomeric formhaving such configuration. Thus, in certain aspects, this inventionrelates to each individual enantiomer or to an enantiomer-enrichedmixture of enantiomers comprising predominately one enantiomer or theother enantiomer. In other embodiments, the compounds of the inventionare present as racemic mixtures of enantiomers.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, that is,where one or more atoms have been enriched with atoms having an atomicmass different from the atomic mass predominately found in nature.Examples of isotopes that may be incorporated into the compounds offormula I, for example, include, but are not limited to, ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ³⁶Cl, and ¹⁸F. Of particular interest arecompounds of formula I enriched in tritium or carbon-14 which can beused, for example, in tissue distribution studies; compounds of formulaI enriched in deuterium especially at a site of metabolism resulting,for example, in compounds having greater metabolic stability; andcompounds of formula I enriched in a positron emitting isotope, such as¹¹C, ¹⁸F, ¹⁵O and ¹³N, which can be used, for example, in PositronEmission Topography (PET) studies.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially available AutoNom software (MDL, San Leandro,Calif.).

REPRESENTATIVE EMBODIMENTS

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

In one aspect, this invention relates to compounds of formula I:

The R¹ moiety is selected from:

H;

—C₁₋₈alkyl, e.g., —CH₃, —CH₂CH₃, —(CH₂)₂CH₃, —CH(CH₃)₂, —C(CH₃)₃,—CH₂CH(CH₃)₂, —(CH₂)₃CH₃, —(CH₂)₄CH₃, —(CH₂)₂CH(CH₃)₂, —(CH₂)₅CH₃, and—(CH₂)₆CH₃;

—C₁₋₃allcylene-C₆₋₁₀aryl, e.g., benzyl;

—C₁₋₃allcylene-C₁₋₉heteroaryl, e.g., —CH₂-pyridinyl and—(CH₂)₂-pyridinyl;

—C₃₋₇cycloalkyl, e.g., cyclopentyl;

—[(CH₂)₂O]₁₋₃CH₃, e.g., —(CH₂)₂OCH₃ and —[(CH₂)₂O]₂CH₃;

—C₁₋₆allylene-OC(O)R¹⁰, e.g., —CH₂OC(O)CH₃, —CH₂OC(O)CH₂CH₃,—CH₂OC(O)(CH₂)₂CH₃, —CH₂CH(CH₃)OC(O)CH₂CH₃, —CH₂OC(O)OCH₃,—CH₂OC(O)OCH₂CH₃, —CH(CH₃)OC(O)OCH₂CH₃, —CH(CH₃)OC(O)O—CH(CH₃)₂,—CH₂CH(CH₃)OC(O)-cyclopentyl, —CH₂OC(O)O-cyclopropyl,—CH(CH₃)—OC(O)—O-cyclohexyl, —CH₂OC(O)O-cyclopentyl,—CH₂CH(CH₃)OC(O)-phenyl, —CH₂OC(O)O-phenyl, —CH₂OC(O)—CH[CH(CH₃)₂]—NH₂,—CH₂OC(O)—CH[CH(CH₃)₂]—NHC(O)OCH₃, and —CH(CH₃)OC(O)—CH(NH₂)CH₂COOCH₃;

—C₁₋₆alkylene-NR¹¹R¹², e.g., —(CH₂)₂—N(CH₃)₂,

—C₁₋₆alkylene-C(O)R¹³, e.g., —CH₂C(O)OCH₃, —CH₂C(O)O-benzyl,—CH₂C(O)—N(CH₃)₂, and

—C₀₋₆alkylenemorpholine, e.g., —(CH₂)₂-morpholine and—(CH₂)₃-morpholine:

—C₁₋₆alkylene-SO₂—C₁₋₆alkyl, e.g., —(CH₂)₂SO₂CH₃;

The R¹⁰ moiety is selected from:

-   -   —C₁₋₆alkyl, e.g., CH₃ and —CH₂CH₃;    -   —O—C₁₋₆alkyl, e.g., —OCH₃, —O—CH₂CH₃, and —O—CH(CH₃)₂;    -   —C₃₋₇cycloalkyl, e.g., cyclopentyl);    -   —O—C₃₋₇cycloalkyl, e.g., —O-cyclopropyl, —O-cyclohexyl, and        —O-cyclopentyl; phenyl;    -   —O-phenyl;    -   —NR¹¹R¹²;    -   —CH(R¹⁵)—NH₂, e.g., —CH[CH(CH₃)₂]—NH₂;    -   —CH(R¹⁵)—NHC(O)O—C₁₋₆alkyl, e.g., —CH[CH(CH₃)₂]—NHC(O)OCH₃; and    -   —CH(NH₂)CH₂COOCH₃.        The R¹¹ and R¹² moieties are independently selected from H,        —C₁₋₆alkyl (e.g., CH₃), and benzyl. Alternately, the R¹¹ and R¹²        moieties can be taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or        —(CH₂)₂O(CH₂)₂—, for example to form a group such as:

The R¹³ moiety is selected from —O—C₁₋₆alkyl, e.g., —OCH₃, —O-benzyl,and —NR¹¹R¹², e.g., —N(CH₃)₂, and

The R¹⁴ moiety is —C₁₋₆alkyl (e.g., —CH₃ and —C(CH₃)₃) or—C₀₋₆alkylene-C₆₋₁₀aryl. The R¹⁵ moiety is H, —CH₃, —CH(CH₃)₂, phenyl,or benzyl.

In addition, each alkyl group in R¹ is optionally substituted with 1 to8 fluoro atoms. For example, when R¹ is —C₁₋₈alkyl, R¹ can also be agroup such as —CH₂CF₃, —CH(CH₃)CF₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃,—CH(CF₃)₂, —CH(CH₂F)₂, —C(CF₃)₂CH₃, and —CH(CH₃)CF₂CF₃.

In one embodiment, R¹ is selected from H, —C₁₋₈alkyl,—C₁₋₆alkylene-OC(O)R¹⁰, and

where R¹⁰ is —C₁₋₆alkyl, —O—C₁₋₆alkyl, or —CH[R¹⁵]—NHC(O)O—C₁₋₆alkyl;R¹⁴ is —C₁₋₆alkyl; R¹⁵ is —CH(CH₃)₂; and each alkyl group in R¹ isoptionally substituted with 1 to 8 fluoro atoms. In one specificembodiment, R¹ is selected from H, —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂,—(CH₂)₃CH₃, —(CH₂)₆CH₃, —CH₂CF₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃,—CH₂OC(O)CH₃, —CH₂OC(O)CH₂CH₃, —CH₂OC(O)(CH₂)₂CH₃, —CH₂OC(O)OCH₂CH₃,—CH₂OC(O)—CH[CH(CH₃)₂]—NHC(O)O—CH₃, and

where R¹⁴ is —CH₃. In other embodiments these compounds have formulasIIa-IId, IIi-IIk, IIIa-IIIb, and IVa-IVd.

In one embodiment, R¹ is H. In other embodiments these compounds haveformulas IIa-IId, IIi-IIk, IIIa-IIIb, and IVa-IVd.

In another embodiment, R¹ is selected from —C₁₋₈alkyl,—C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl,—[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹⁰, —C₁₋₆alkylene-NR¹¹R¹²,—C₁₋₆alkylene-C(O)R¹³, —C₀₋₆alkylenemorpholinyl,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

In other embodiments these compounds have formulas IIa-IId, IIi-IIk,IIIa-IIIb, and IVa-IVd. In one aspect of the invention, these compoundsmay find particular utility as prodrugs or as intermediates in thesynthetic procedures described herein. Specific examples of such prodrugmoieties include where R¹ is —C₁₋₆alkylene-OC(O)R¹⁰, such as—CH(CH₃)OC(O)—O-cyclohexyl:

making the compound a cilexetil ester; or R¹ is —C₀₋₆alkylenemorpholinesuch as (CH₂)₂-morpholine:

making the compound a 2-morpholinoethyl or mofetil ester; or

such as —CH₂-5-methyl-[1,3]dioxol-2-one:

making the compound a medoxomil ester.

In one embodiment, R² is —OR²¹ or —CH₂OR²¹, and R³ is H or —CH₃. Theseembodiments can be depicted as formulas IIa-IId:

The R²¹ moiety is H, —C(O)—C₁₋₆alkyl, —C(O)—CH(R²²)—NH₂,—C(O)—CH(R²²)—NHC(O)O—C₁₋₆alkyl, or —P(O)(OR²³)₂; and in one particularembodiment, R²¹ moiety is H. The R²² moiety is H, —CH₃, —CH(CH₃)₂,phenyl, or benzyl. The R²³ moiety is H, —C₁₋₆alkyl, or phenyl.

In one embodiment, compounds of the invention have formula Ha, and inone exemplary embodiment, R¹ is selected from H, —C₁₋₈alkyl,—C₁₋₆alkylene-OC(O)R¹⁶, and

where R¹⁰ is —C₁₋₆alkyl, —O—C₁₋₆alkyl, or —CH[R¹⁵]—NHC(O)O—C₁₋₆alkyl;R¹⁴ is —C₁₋₆alkyl; R¹⁵ is —CH(CH₃)₂; and each alkyl group in R¹ isoptionally substituted with 1 to 8 fluoro atoms; Z is selected from —CH—and —N—; R⁴ is selected from H, —C₁₋₈alkyl, —C₁₋₃alkylene-O—C₁₋₈alkyl,—C₁₋₃alkylene-O—C₆₋₁₀aryl, —[(CH₂)₂O]₁₋₃CH₃, and

where R⁴⁴ is —C₁₋₆alkyl; and each alkyl group in R⁴ is optionallysubstituted with 1 to 8 fluoro atoms; a is 0 and b is 0; or a is 0, b is1, and R⁶ is halo; or a is 0, b is 2, and one R⁶ is halo and the otherR⁶ is halo or —CH₃; or a is 1, R⁵ is halo, and b is 0; or a is 1, R⁵ ishalo, b is 1, and R⁶ is halo; or a is 1, R⁵ is halo, b is 2, and each R⁶is halo; and where the methylene linker on the biphenyl is optionallysubstituted with two —CH₃ groups; and in another exemplary embodiment,R¹ is selected from H, —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —(CH₂)₃CH₃,—(CH₂)₆CH₃, —CH₂CF₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃, —CH₂OC(O)CH₃,—CH₂OC(O)CH₂CH₃, —CH₂OC(O)(CH₂)₂CH₃, —CH₂OC(O)OCH₂CH₃,—CH₂OC(O)—CH[CH(CH₃)₂]—NHC(O)O—CH₃, and

where R¹⁴ is —CH₃; R⁴ is selected from H, —CH₂CH₃, —CH(CH₃)₂,—CH₂CH(CH₃)₂, —(CH₂)₃CH₃, —C(CH₃)₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃,—(CH₂)₃—O—CH₂CH₃, —(CH₂)₂—O- phenyl, —(CH₂)₂OCH₃, and

where R⁴⁴ is —CH₃; and a is 0 and b is 0; or a is 0, b is 1, and R⁶ is2′-fluoro, 3′-fluoro, 3′-chloro, or 4′-flouro; or a is 0, b is 2, and R⁶is 2′-fluoro, 5′-chloro or 2′-methyl, 5′-chloro or 2′,5′-dichloro; or ais 1, R⁵ is 3-chloro, and b is 0; or a is 1, R⁵ is 3-chloro, b is 1, andR⁶ is 3′-chloro; or a is 1, R⁵ is 3-chloro, b is 2, and R⁶ is 2′-fluoro,5′-chloro.

In one embodiment, compounds of the invention have formula IIb, and inone exemplary embodiment, H or —C₁₋₈alkyl; Z is —N—; R⁴ is H or—C₁₋₈alkyl; and a and b are 0; and in another exemplary embodiment, R¹and R⁴ are H.

In one embodiment, compounds of the invention have formula IIc, and inone exemplary embodiment, R¹ is H or —C₁₋₈alkyl; Z is —CH—; R⁴ is H or—C₁₋₈alkyl; a is 0 or a is 1 and R⁵ is halo; b is 0 or b is 1 or 2 andR⁶ is halo; and where the methylene linker on the biphenyl is optionallysubstituted with two —CH₃ groups; and in another exemplary embodiment,R¹ is H, —CH₂CH₃, or —(CH₂)₃CH₃; R⁴ is H; a is 0 or a is 1 and R⁵ is3-chloro; b is 0 or b is 1 and R⁶ is 2′-fluoro, 3′-fluoro, 3′-chloro, or4′-flouro.

In one embodiment, compounds of the invention have formula IId, and inone exemplary embodiment, R¹ is H or —C₁₋₈alkyl; Z is —CH—; R⁴ is H or—C₁₋₈alkyl; a is 0; and b is 0, or b is 1 and R⁶ is halo; and in anotherexemplary embodiment, R¹ is H or —CH₂CH₃; R⁴ is H or —CH₂CH(CH₃)₂; and bis 0, or b is 1 and R⁶ is 2′-fluoro, 3′-fluoro, 3′-chloro, or 4′-flouro.

In another embodiment, R² is taken together with R¹ to form —OCR¹⁵R¹⁶—or —CH₂O—CR¹⁵R¹⁶—, and R³ is selected from H and —CH₃. The R¹⁵ and R¹⁶moieties are independently selected from H, —C₁₋₆alkyl, and—O—C₃₋₂cycloalkyl, or R¹⁵ and R¹⁶ are taken together to form ═O. Thesecan be depicted as formulas IIe-IIh:

In one aspect of the invention, these compounds may find particularutility as prodrugs or as intermediates in the synthetic proceduresdescribed herein. Compounds where R² is —CH₂OP(O)(OH)₂ may also findutility as prodrugs. In one embodiment of the compounds of formulas IIe,IIf, IIg, and IIh, Z is —CH—, R⁴ is H, a is 0, b is 1, R⁶ is 3′Cl, andR¹⁵ and R¹⁶ are H.

In another embodiment, R² is taken together with R³ to form —CH₂—O—CH₂—or —CH₂—CH₂—, which can be depicted as formulas IIi and IIj,respectively:

In another embodiment, R² and R³ are both —CH₃, which can be depicted asformula IIk:

In one embodiment of the compounds of formulas IIi, IIj, and IIk, R¹ isH, Z is —CH—, R⁴ is —C₁₋₈alkyl (e.g., —CH₂CH(CH₃)₂), a is 0, b is 1, andR⁶ is 3′Cl.

The Z group is selected from —CH— and —N—. These embodiments can bedepicted as formulas IIIa and IIIb:

The R⁴ moiety is selected from:

H;

—C₁₋₈alkyl, e.g., —CH₃, —CH₂CH₃, —(CH₂)₂CH₃, —CH(CH₃)₂, —C(CH₃)₃,—CH₂CH(CH₃)₂, —(CH₂)₃CH₃, —(CH₂)₄CH₃, —(CH₂)₂CH(CH₃)₂, —(CH₂)₅CH₃, and—(CH₂)₆CH₃;

—C₁₋₃alkylene-O—C₁₋₈alkyl e.g., —(CH₂)₃—O—CH₂CH₃;

—C₁₋₃alkylene-C₆₋₁₀aryl, e.g., benzyl;

—C₁₋₃alkylene-O—C₆₋₁₀aryl, e.g., —(CH₂)₂—O-phenyl;

—C₁₋₃ alkylene-C₁₋₉heteroaryl, e.g., —CH₂-pyridinyl and—(CH₂)₂-pyridinyl;

—C₃₋₂cycloalkyl, e.g., cyclopentyl;

—[(CH₂)₂O]₁₋₃CH₃, e.g., —(CH₂)₂OCH₃ and —[(CH₂)₂O]₂CH₃;—C₁₋₆allylene-OC(O)R⁴⁰, e.g., —CH₂OC(O)CH₃, —CH₂OC(O)CH₂CH₃,—CH₂OC(O)(CH₂)₂CH₃, —CH₂CH(CH₃)OC(O)CH₂CH₃, —CH₂OC(O)OCH₃,—CH₂OC(O)OCH₂CH₃, —CH(CH₃)OC(O)OCH₂CH₃, —CH(CH₃)OC(O)O—CH(CH₃)₂,—CH₂CH(CH₃)OC(O)-cyclopentyl, —CH₂OC(O)O-cyclopropyl, —CH(CH₃)—OC(O)—O—cyclohexyl, —CH₂OC(O)O-cyclopentyl, —CH₂CH(CH₃)OC(O)-phenyl, —CH₂OC(O)O—phenyl, —CH₂OC(O)—CH[CH(CH₃)₂]—NH₂, —CH₂OC(O)—CH[CH(CH₃)₂]—NHC(O)OCH₃,and —CH(CH₃)OC(O)—CH(NH₂)CH₂COOCH₃;

—C₁₋₆alkylene-NR⁴¹R⁴², e.g., —(CH₂)₂—N(CH₃)₂,

—C₁₋₆alkylene-C(O)R⁴³, e.g., —CH₂C(O)OCH₃, —CH₂C(O)O-benzyl,—CH₂C(O)—N(CH₃)₂, and

—C₀₋₆alkylenemorpholine, e.g., —(CH₂)₂-morpholine and—(CH₂)₃-morpholine:

—C₁₋₆alkylene-SO₂—C₁₋₆alkyl, e.g., —(CH₂)₂SO₂CH₃;

The R⁴⁰ moiety is selected from:

—C₁₋₆alkyl, e.g., CH₃ and —CH₂CH₃;

—O—C₁₋₆alkyl, e.g., —OCH₃, —O—CH₂CH₃, and —O—CH(CH₃)₂;

—C₃₋₇cycloalkyl, e.g., cyclopentyl;

—O—C₃₋₇cycloalkyl, e.g., —O-cyclopropyl, —O-cyclohexyl, and—O-cyclopentyl; phenyl;

—O-phenyl;

—NR⁴¹R⁴²;

—CH(R⁴⁵)—NH₂, e.g., —CH[CH(CH₃)₂]—NH₂;

—CH(R⁴⁵)—NHC(O)O—C₁₋₆alkyl, e.g., —CH[CH(CH₃)₂]—NHC(O)OCH₃; and

—CH(NH₂)CH₂COOCH₃.

The R⁴¹ and R⁴² moieties are independently selected from H, —C₁₋₆alkyl(e.g., CH₃), and benzyl. Alternately, the R⁴¹ and R⁴² moieties can betaken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—, forexample to form a group such as:

The R⁴³ moiety is selected from —O—C₁₋₆alkyl, e.g., —OCH₃, —O-benzyl,and —NR⁴¹R⁴². e.g., —N(CH₃)₂, and

The R⁴⁴ moiety is —C₁₋₆alkyl (e.g., —CH₃ and —C(CH₃)₃) or—C₀₋₆alkylene-C₆₋₁₀aryl. The R⁴⁵ moiety is H, —CH₃, —CH(CH₃)₂, phenyl,or benzyl.

In addition, each alkyl group in R⁴ is optionally substituted with 1 to8 fluoro atoms. For example, when R⁴ is —C₁₋₈alkyl, R⁴ can also be agroup such as —CH₂CF₃, —CH(CH₃)CF₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃,—CH(CF₃)₂, —CH(CH₂F)₂, —C(CF₃)₂CH₃, and —CH(CH₃)CF₂CF₃.

In one embodiment, R⁴ is selected from H, —C₁₋₈alkyl,—C₁₋₃alkylene-O—C₁₋₈alkyl, —C₁₋₃alkylene-O—C₆₋₁₀aryl, —[(CH₂)₂O]₁₋₃CH₃,and

where R⁴⁴ is —C₁₋₆alkyl; and each alkyl group in R⁴ is optionallysubstituted with 1 to 8 fluoro atoms. In one specific embodiment, R⁴ isselected from H, —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —(CH₂)₃CH₃, —C(CH₃)₃,—(CH₂)₂CF₃, —CH₂CF₂CH₃, —(CH₂)₃—O—CH₂CH₃, —(CH₂)₂—O-phenyl, —(CH₂)₂OCH₃,and

where R⁴⁴ is —CH₃. In other embodiments these compounds have formulasIIa-IIk, IIIa-IIIb, and IVa-IVd.

In one embodiment, R¹ is H. In other embodiments these compounds haveformulas IIa-IIk, IIIa-IIIb, and IVa-IVd. In yet another embodiment,both R¹ and R⁴ are H. In other embodiments these compounds have formulasIIa-IIh, IIm-IIo, IIIa-IIIb, and IVa-IVd.

In another embodiment, R⁴ is selected from —C₁₋₈alkyl,—C₁₋₃alkylene-O—C₁₋₈alkyl, —C₁₋₃ alkylene-C₆₋₁₀aryl,—C₁₋₃alkylene-O—C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl,—C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R⁴⁰,—C₁₋₆alkylene-NR⁴¹R⁴², —C₁₋₆alkylene-C(O)R⁴³, —C₀₋₆alkylenemorpholinyl,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

In other embodiments these compounds have formulas IIa-IIk, IIIa-IIIb,and IVa-IVd. In one aspect of the invention, these compounds may findparticular utility as prodrugs or as intermediates in the syntheticprocedures described herein. In one embodiment, both R¹ and R⁴ are suchprodrug moieties. In another embodiment, one of R¹ and R⁴ is a prodrugmoiety and the other is H. Specific examples of such prodrug moietiesinclude where R⁴ is —C₁₋₆alkylene-OC(O)R¹⁰, such asCH(CH₃)OC(O)—O-cyclohexyl:

making the compound a cilexetil ester; or R⁴ is —C₀₋₆alkylenemorpholinesuch as (CH₂)₂-morpholine:

making the compound a 2-morpholinoethyl or mofetil ester; or

such as —CH₂-5-methyl-[1,3]dioxol-2-one:

making the compound a medoxomil ester.

The numbering for the R⁵ and R⁶ groups is as follows:

The integer “a” is 0 or 1. The R⁵ moiety, when present, is selected fromhalo, —CH₃, —CF₃, and —CN. In one embodiment, a is 0. In anotherembodiment, a is 1, and R⁵ is halo, such as 3-chloro or 3-fluoro. In yetanother embodiment a is 0, or a is 1 and R⁵ is halo. In otherembodiments these compounds have formulas IIa-IIk, IIIa-IIIb, andIVa-IVd.

The integer “b” is 0 or an integer from 1 to 3. The R⁶ moiety, whenpresent, is independently selected from halo, —OH, —CH₃, —OCH₃, —CN, and—CF₃. In one embodiment, b is 0. In another embodiment, b is 1 and R⁶ isselected from Cl, F, —OH, —CH₃, —OCH₃, —CN, and —CF₃, such 2′-chloro,3′-chloro, 2′-fluoro, 3′-fluoro, 2′-hydroxy, 3′-hydroxy, 3′-methyl,2′-methoxy, 3′-cyano, or 3′-trifluoromethyl. In another embodiment, b is1 and R⁶ is halo, —CH₃, or —OCH₃, such 3′-chloro, 3′-methyl, or2′-methoxy. In another embodiment, b is 2 and R⁶ is 2′-fluoro-5′-chloro,2′,5′-dichloro, 2′,5′-difluoro, 2′-methyl-5′-chloro,3′-fluoro-5′-chloro, 3′-hydroxy-5′-chloro, 3′,5′-dichloro,3′,5′-difluoro, 2′-methoxy-5′-chloro, 2′-methoxy-5′-fluoro,2′-hydroxy-5′-fluoro, 2′-fluoro-3′-chloro, 2′-hydroxy-5′-chloro, or2′-hydroxy-3′-chloro. In another embodiment, b is 3 and each R⁶ isindependently halo or —CH₃, such as 2′-methyl-3′,5′-dichloro or2′-fluoro-3′-methyl-5′-chloro. In one particular embodiment, b is 0, orb is 1 and R⁶ is halo, or b is 2 and each R⁶ is independently selectedfrom halo and —CH₃. In other embodiments these compounds have formulasIIa-IIk, IIIa-IIIb, and IVa-IVd.

In other exemplary embodiments, a is 0 and b is 0; or a is 0, b is 1,and R⁶ is 2′-fluoro, 3′-fluoro, 3′-chloro, or 4′-flouro; or a is 0, b is2, and R⁶ is 2′-fluoro, 5′-chloro or 2′-methyl, 5′-chloro or2′,5′-dichloro; a is 1, R⁵ is 3-chloro, and b is 0; or a is 1, R⁵ is3-chloro, b is 1, and R⁶ is 3′-chloro; or a is 1, R⁵ is 3-chloro, b is2, and R⁶ is 2′-fluoro, 5′-chloro. In other embodiments these compoundshave formulas IIa-IIk, IIIa-IIIb, and IVa-IVd. Of particular interestare compounds of the formulas:

The methylene linker on the biphenyl is optionally substituted with oneor two —C₁₋₆ alkyl groups or cyclopropyl. For example, in oneembodiment, the methylene linker on the biphenyl is unsubstituted; inanother embodiment, the methylene linker on the biphenyl is substitutedwith one —C₁₋₆alkyl group (e.g., —CH₃); and in yet another embodiment,the methylene linker on the biphenyl is substituted with two —C₁₋₆alkylgroups (e.g., two —CH₃ groups); in another embodiment, the methylenelinker on the biphenyl is substituted with a cyclopropyl group. Theseembodiments are depicted, respectively, as formulas IVa-IVd:

In one embodiment of the compounds of formulas IVa, IVb, IVc, and IVd,R¹ is H, R² is —OR²¹ and R²¹ is H, R³ is H, Z is —CH—, R⁴ is —C₁₋₈alkyl(e.g., —CH₂CH(CH₃)₂), a is 0, b is 1, and R⁶ is 3′Cl.

In another embodiment, R¹ is selected from H, —C₁₋₈alkyl,—C₁₋₆alkylene-OC(O)R¹⁶, and

where R¹⁰ is —C₁₋₆alkyl, —O—C₁₋₆alkyl, or —CH[R¹⁵]—NHC(O)O—C₁₋₆alkyl;R¹⁴ is —C₁₋₆alkyl; R¹⁵ is —CH(CH₃)₂; and each alkyl group in R¹ isoptionally substituted with 1 to 8 fluoro atoms;

R⁴ is selected from H, —C₁₋₈alkyl, —C₁₋₃alkylene-O—C₁₋₈alkyl,—C₁₋₃alkylene-O—C₆₋₁₀aryl, —[(CH₂)₂O]₁₋₃CH₃, and

where R⁴⁴ is —C₁₋₆alkyl; and each alkyl group in R⁴ is optionallysubstituted with 1 to 8 fluoro atoms;

a is 0 and b is 0; or a is 0, b is 1, and R⁶ is 2′-fluoro, 3′-fluoro,3′-chloro, or 4′-flouro; or a is 0, b is 2, and R⁶ is 2′-fluoro,5′-chloro or 2′-methyl, 5′-chloro or 2′,5′-dichloro; or a is 1, R⁵ is3-chloro, and b is 0; or a is 1, R⁵ is 3-chloro, b is 1, and R⁶ is3′-chloro; or a is 1, R⁵ is 3-chloro, b is 2, and R⁶ is 2′-fluoro,5′-chloro; and where the methylene linker on the biphenyl is optionallysubstituted with two —CH₃ groups. In one particular embodiment of thesecompounds, R² is —OR²¹ or —CH₂OR²¹; and R³ is H or —CH₃; where R²¹ is H.

In addition, particular compounds of formula I that are of interestinclude those set forth in the Examples below, as well aspharmaceutically acceptable salts thereof.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (for example, reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. In some instances, reactions were conducted at roomtemperature and no actual temperature measurement was taken. It isunderstood that room temperature can be taken to mean a temperaturewithin the range commonly associated with the ambient temperature in alaboratory environment, and will typically be in the range of about 18°C. to about 30° C. In other instances, reactions were conducted at roomtemperature and the temperature was actually measured and recorded.While optimum reaction conditions will typically vary depending onvarious reaction parameters such as the particular reactants, solventsand quantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Protecting groups other than those illustrated in the proceduresdescribed herein may be used, if desired. For example, numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis,Fourth Edition, Wiley, New York, 2006, and references cited therein.

Carboxy-protecting groups are suitable for preventing undesiredreactions at a carboxy group, and examples include, but are not limitedto, methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), trimethylsilyl (TMS), t-butyldimethylsilyl(TBDMS), diphenylmethyl (benzhydryl, DPM) and the like. Amino-protectinggroups are suitable for preventing undesired reactions at an aminogroup, and examples include, but are not limited to, t-butoxycarbonyl(BOC), trityl (Tr), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl(Fmoc), formyl, trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), andthe like. Hydroxyl-protecting groups are suitable for preventingundesired reactions at a hydroxyl group, and examples include, but arenot limited to C₁₋₆alkyls, say′ groups including triC₁₋₆alkylsilylgroups, such as trimethylsilyl (TMS), triethylsilyl (TES), andt-butyldimethylsilyl (TBDMS); esters (acyl groups) includingC₁₋₆alkanoyl groups, such as formyl, acetyl, and pivaloyl, and aromaticacyl groups such as benzoyl; arylmethyl groups such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); and the like.

Standard deprotection techniques and reagents are used to remove theprotecting groups, and may vary depending upon which group is used. Forexample, sodium or lithium hydroxide is commonly used when thecarboxy-protecting group is methyl, an acid such as TFA or HCl iscommonly used when the carboxy-protecting group is ethyl or t-butyl, andH₂/Pd/C may be used when the carboxy-protecting group is benzyl. A BOCamino-protecting group can be removed using an acidic reagent such asTFA in DCM or HCl in 1,4-dioxane, while a Cbz amino-protecting group canbe removed by employing catalytic hydrogenation conditions such as H₂ (1atm) and 10% Pd/C in an alcoholic solvent (“H₂/Pd/C”). H₂/Pd/C iscommonly used when the hydroxyl-protecting group is benzyl, while NaOHis commonly used when the hydroxyl-protecting group is an acyl group.

Suitable bases for use in these schemes include, by way of illustrationand not limitation, potassium carbonate, calcium carbonate, sodiumcarbonate, triethylamine, pyridine, 1,8-diazabicyclo-[5.4.0]undec-7-ene(DBU), N, N-diisopropylethylamine (DIPEA), 4-methylmorpholine, sodiumhydroxide, potassium hydroxide, potassium t-butoxide, and metalhydrides.

Suitable inert diluents or solvents for use in these schemes include, byway of illustration and not limitation, tetrahydrofuran (THF),acetonitrile (MeCN), N,N-dimethylformamide (DMF), N,N-dimethylacetamide(DMA), dimethyl sulfoxide (DMSO), toluene, dichloromethane (DCM),chloroform (CHCl₃), carbon tetrachloride (CCl₄), 1,4-dioxane, methanol,ethanol, water, and the like.

Suitable carboxylic acid/amine coupling reagents includebenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), N,N,N; N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU),(2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate) (HCTU), 1,3-dicyclohexylcarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI),carbonyldiimidazole (CDI), 1-hydroxybenzotriazole (HOBt), and the like.Coupling reactions are conducted in an inert diluent in the presence ofa base such as DIPEA, and are performed under conventional amidebond-forming conditions.

All reactions are typically conducted at a temperature within the rangeof about −78 C to 100° C., for example at room temperature. Reactionsmay be monitored by use of thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and/or LCMS until completion.Reactions may be complete in minutes, or may take hours, typically from1-2 hours and up to 48 hours. Upon completion, the resulting mixture orreaction product may be further treated in order to obtain the desiredproduct. For example, the resulting mixture or reaction product may besubjected to one or more of the following procedures: concentrating orpartitioning (for example, between EtOAc and water or between 5% THF inEtOAc and 1M phosphoric acid); extraction (for example, with EtOAc,CHCl₃, DCM, chloroform); washing (for example, with saturated aqueousNaCl, saturated aqueous NaHCO₃, Na₂CO₃ (5%), CHCl₃ or 1M NaOH); drying(for example, over MgSO₄, over Na₂SO₄, or in vacuo); filtering;crystallizing (for example, from EtOAc and hexanes); being concentrated(for example, in vacuo); and/or purification (e.g., silica gelchromatography, flash chromatography, preparative HPLC, reversephase-HPLC, or crystallization).

Compounds of formula I, as well as their salts, can be prepared as shownin Scheme I:

The process comprises the step of coupling compound 1 with compound 2,where R¹-R⁶, Z, a, and b are as defined for formula I, and P is selectedfrom H and a suitable amino-protecting group, examples of which includet-butoxycarbonyl, trityl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl,formyl, trimethylsilyl, and t-butyldimethylsilyl. When P is an aminoprotecting group, the process further comprises deprotecting thecompound of formula 1, before or in situ with the coupling step.

In instances where R¹ is a group such as —OCH₃ or OCH₂CH₃, the couplingstep may be followed by a deprotection step to provide a compound offormula I where R¹ is a group such as —OH. Thus, one method of preparingcompounds of the invention involves coupling compounds 1 and 2, with anoptional deprotection step to form a compound of formula I or apharmaceutically acceptable salt thereof.

Methods of preparing compound 1 are described in the Examples. Compound2 is generally commercially available or can be prepared usingprocedures that are known in the art.

Certain intermediates described herein are believed to be novel andaccordingly, such compounds are provided as further aspects of theinvention including, for example, the compounds of formula 1, or a saltthereof:

where P is H or a suitable amino-protecting group, examples of whichinclude, t-butoxycarbonyl, trityl, benzyloxycarbonyl,9-fluorenylmethoxycarbonyl, formyl, trimethylsilyl, andt-butyldimethylsilyl; and R¹, R², R³, R⁵, R⁶, Z, a and b are as definedfor formula I.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Utility

Compounds of the invention possess neprilysin (NEP) inhibition activity,that is, the compounds are able to inhibit enzyme-catalytic activity. Inanother embodiment, the compounds do not exhibit significant inhibitoryactivity of the angiotensin-converting enzyme. One measure of theability of a compound to inhibit NEP activity is the inhibition constant(pK_(i)). The pK_(i) value is the negative logarithm to base 10 of thedissociation constant (K_(i)), which is typically reported in molarunits. Compounds of the invention of particular interest are thosehaving a pK_(i) at NEP greater than or equal to 6.0, particularly thosehaving a pK_(i) greater than or equal to 7.0, and even more particularlythose having a pK_(i) greater than or equal to 8.0. In one embodiment,compounds of interest have a pK_(i) in the range of 6.0-6.9; in anotherembodiment, compounds of interest have a pK_(i) in the range of 7.0-7.9;in yet another embodiment, compounds of interest have a pK_(i) in therange of 8.0-8.9; and in still another embodiment, compounds of interesthave a pK_(i) in the range of greater than or equal to 9.0. Such valuescan be determined by techniques that are well known in the art, as wellas in the assays described herein.

Another measure of the ability of a compound to inhibit NEP activity isthe apparent inhibition constant (IC₅₀), which is the molarconcentration of compound that results in half-maximal inhibition ofsubstrate conversion by the NEP enzyme. The pIC₅₀ value is the negativelogarithm to base 10 of the IC₅₀. Compounds of the invention that are ofparticular interest, include those that exhibit a pIC₅₀ for NEP greaterthan or equal to about 5.0. Compounds of interest also include thosehaving a pIC₅₀ for NEP ≧about 6.0 or a pIC₅₀ for NEP ≧about 7.0. Inanother embodiment, compounds of interest have a pIC₅₀ for NEP withinthe range of about 7.0-11.0; and in another embodiment, within the rangeof about 8.0-11.0, such as within the range of about 8.0-10.0.

It is noted that in some cases, compounds of the invention may possessweak NEP inhibition activity. In such cases, those of skill in the artwill recognize that these compounds still have utility as researchtools.

Exemplary assays to determine properties of compounds of the invention,such as the NEP inhibiting activity, are described in the Examples andinclude by way of illustration and not limitation, assays that measureNEP inhibition (described in Assay 1). Useful secondary assays includeassays to measure ACE inhibition (also described in Assay 1) andaminopeptidase P (APP) inhibition (described in Sulpizio et al. (2005)JPET 315:1306-1313). A pharmacodynamic assay to assess the in vivoinhibitory potencies for ACE and NEP in anesthetized rats is describedin Assay 2 (see also Seymour et al. (1985) Hypertension 7(SupplI):I-35-I-42 and Wigle et al. (1992) Can. J. Physiol. Pharmacol.70:1525-1528), where ACE inhibition is measured as the percentinhibition of the angiotensin I pressor response and NEP inhibition ismeasured as increased urinary cyclic guanosine 3′,5′-monophosphate(cGMP) output.

There are many in vivo assays that can be used to ascertain furtherutilities of the compounds of the invention. The conscious spontaneouslyhypertensive rat (SHR) model is a renin dependent hypertension model,and is described in Assay 3. See also Intengan et al. (1999) Circulation100(22):2267-2275 and Badyal et al. (2003) Indian Journal ofPharmacology 35:349-362. The conscious desoxycorticosterone acetate-salt(DOCA-salt) rat model is a volume dependent hypertension model that isuseful for measuring NEP activity, and is described in Assay 4. See alsoTrapani et al. (1989) J. Cardiovasc. Pharmacol. 14:419-424, Intengan etal. (1999) Hypertension 34(4):907-913, and Badyal et al. (2003) supra).The DOCA-salt model is particularly useful for evaluating the ability ofa test compound to reduce blood pressure as well as to measure a testcompound's ability to prevent or delay a rise in blood pressure. TheDahl salt-sensive (DSS) hypertensive rat model is a model ofhypertension that is sensitive to dietary salt (NaCl), and is describedin Assay 5. See also Rapp (1982) Hypertension 4:753-763. The ratmonocrotaline model of pulmonary arterial hypertension described, forexample, in Kato et al. (2008) J. Cardiovasc. Pharmacol. 51(1):18-23, isa reliable predictor of clinical efficacy for the treatment of pulmonaryarterial hypertension. Heart failure animal models include the DSS ratmodel for heart failure and the aorto-caval fistula model (AV shunt),the latter of which is described, for example, in Norling et al. (1996)J. Amer. Soc. Nephrol. 7:1038-1044. Other animal models, such as the hotplate, tail-flick and formalin tests, can be used to measure theanalgesic properties of compounds of the invention, as well as thespinal nerve ligation (SNL) model of neuropathic pain. See, for example,Malmberg et al. (1999) Current Protocols in Neuroscience 8.9.1-8.9.15.

Compounds of the invention are expected to inhibit the NEP enzyme in anyof the assays listed above, or assays of a similar nature. Thus, theaforementioned assays are useful in determining the therapeutic utilityof compounds of the invention, for example, their utility asantihypertensive agents or antidiarrheal agents. Other properties andutilities of compounds of the invention can be demonstrated using otherin vitro and in vivo assays well-known to those skilled in the art.Compounds of formula I may be active drugs as well as prodrugs. Thus,when discussing the activity of compounds of the invention, it isunderstood that any such prodrugs may not exhibit the expected activityin an assay, but are expected to exhibit the desired activity oncemetabolized.

Compounds of the invention are expected to be useful for the treatmentand/or prevention of medical conditions responsive to NEP inhibition.Thus it is expected that patients suffering from a disease or disorderthat is treated by inhibiting the NEP enzyme or by increasing the levelsof its peptide substrates, can be treated by administering atherapeutically effective amount of a compound of the invention. Forexample, by inhibiting NEP, the compounds are expected to potentiate thebiological effects of endogenous peptides that are metabolized by NEP,such as the natriuretic peptides, bombesin, bradykinins, calcitonin,endothelins, enkephalins, neurotensin, substance P and vasoactiveintestinal peptide. Thus, these compounds are expected to have otherphysiological actions, for example, on the renal, central nervous,reproductive and gastrointestinal systems.

In one embodiment of the invention, patients suffering from a disease ordisorder that is treated by inhibiting the NEP enzyme, are treated byadministering a compound of the invention that is in its active form,i.e., a compound of formula I where R¹ and R⁴ are H, and R², R³, R⁵, R⁶,a, b, and Z are as defined for formula I.

In another embodiment, patients are treated by administering a compoundthat is metabolized in vitro to form a compound of formula I where R¹and R⁴ are H, and R², R³, R⁵, R⁶, a, b, and Z are as defined for formulaI.

In another embodiment, patients are treated by administering a compoundof the invention that is in its prodrug form at the R¹ group, i.e., acompound of formula I where R¹ is selected from —C₁₋₈alkyl,—C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl,—[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹⁰, —C₁₋₆alkylene-NR¹¹R¹²,—C₁₋₆alkylene-C(O)R¹³, —C₀₋₆alkylenemorpholinyl,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

In yet another embodiment, patients are treated by administering acompound of the invention that is in its prodrug form at the R⁴ group,i.e., a compound of formula I where R⁴ is selected from —C₁₋₈alkyl,—C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl,—[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R⁴⁰, —C₁₋₆alkylene-NR⁴¹R⁴²,—C₁₋₆alkylene-C(O)R⁴³, —C₀₋₆alkylenemorpholinyl,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

In still another embodiment, patients are treated by administering acompound of the invention that is in its prodrug form at the R¹ groupand at the R⁴ group.

Cardiovascular Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected to findutility in treating and/or preventing medical conditions such ascardiovascular diseases. See, for example, Rogues et al. (1993)Pharmacol. Rev. 45:87-146 and Dempsey et al. (2009) Amer. J. ofPathology 174(3):782-796. Cardiovascular diseases of particular interestinclude hypertension and heart failure. Hypertension includes, by way ofillustration and not limitation: primary hypertension, which is alsoreferred to as essential hypertension or idiopathic hypertension;secondary hypertension; hypertension with accompanying renal disease;severe hypertension with or without accompanying renal disease;pulmonary hypertension, including pulmonary arterial hypertension; andresistant hypertension. Heart failure includes, by way of illustrationand not limitation: congestive heart failure; acute heart failure;chronic heart failure, for example with reduced left ventricularejection fraction (also referred to as systolic heart failure) or withpreserved left ventricular ejection fraction (also referred to asdiastolic heart failure); and acute and chronic decompensated heartfailure, with or without accompanying renal disease. Thus, oneembodiment of the invention relates to a method for treatinghypertension, particularly primary hypertension or pulmonary arterialhypertension, comprising administering to a patient a therapeuticallyeffective amount of a compound of the invention.

For treatment of primary hypertension, the therapeutically effectiveamount is typically the amount that is sufficient to lower the patient'sblood pressure. This would include both mild-to-moderate hypertensionand severe hypertension. When used to treat hypertension, the compoundmay be administered in combination with other therapeutic agents such asaldosterone antagonists, aldosterone synthase inhibitors,angiotensin-converting enzyme inhibitors and dual-actingangiotensin-converting enzyme/neprilysin inhibitors,angiotensin-converting enzyme 2 (ACE2) activators and stimulators,angiotensin-II vaccines, anti-diabetic agents, anti-lipid agents,anti-thrombotic agents, AT₁ receptor antagonists and dual-acting AT₁receptor antagonist/neprilysin inhibitors, β₁-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptorantagonists, calcium channel blockers, diuretics, endothelin receptorantagonists, endothelin converting enzyme inhibitors, neprilysininhibitors, natriuretic peptides and their analogs, natriuretic peptideclearance receptor antagonists, nitric oxide donors, non-steroidalanti-inflammatory agents, phosphodiesterase inhibitors (specificallyPDE-V inhibitors), prostaglandin receptor agonists, renin inhibitors,soluble guanylate cyclase stimulators and activators, and combinationsthereof. In one particular embodiment of the invention, a compound ofthe invention is combined with an AT₁ receptor antagonist, a calciumchannel blocker, a diuretic, or a combination thereof, and used to treatprimary hypertension. In another particular embodiment of the invention,a compound of the invention is combined with an AT₁ receptor antagonist,and used to treat hypertension with accompanying renal disease. Whenused to treat resistant hypertension, the compound may be administeredin combination with other therapeutic agents such as aldosteronesynthase inhibitors.

For treatment of pulmonary arterial hypertension, the therapeuticallyeffective amount is typically the amount that is sufficient to lower thepulmonary vascular resistance. Other goals of therapy are to improve apatient's exercise capacity. For example, in a clinical setting, thetherapeutically effective amount can be the amount that improves apatient's ability to walk comfortably for a period of 6 minutes(covering a distance of approximately 20-40 meters). When used to treatpulmonary arterial hypertension the compound may be administered incombination with other therapeutic agents such as α-adrenergic receptorantagonists, β₁-adrenergic receptor antagonists, β₂-adrenergic receptoragonists, angiotensin-converting enzyme inhibitors, anticoagulants,calcium channel blockers, diuretics, endothelin receptor antagonists,PDE-V inhibitors, prostaglandin analogs, selective serotonin reuptakeinhibitors, and combinations thereof. In one particular embodiment ofthe invention, a compound of the invention is combined with a PDE-Vinhibitor or a selective serotonin reuptake inhibitor and used to treatpulmonary arterial hypertension.

Another embodiment of the invention relates to a method for treatingheart failure, in particular congestive heart failure (including bothsystolic and diastolic congestive heart failure), comprisingadministering to a patient a therapeutically effective amount of acompound of the invention. Typically, the therapeutically effectiveamount is the amount that is sufficient to lower blood pressure and/orimprove renal functions. In a clinical setting, the therapeuticallyeffective amount can be the amount that is sufficient to improve cardiachemodynamics, like for instance reduction in wedge pressure, rightatrial pressure, filling pressure, and vascular resistance. In oneembodiment, the compound is administered as an intravenous dosage form.When used to treat heart failure, the compound may be administered incombination with other therapeutic agents such as adenosine receptorantagonists, advanced glycation end product breakers, aldosteroneantagonists, AT₁ receptor antagonists, β₁-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptorantagonists, chymase inhibitors, digoxin, diuretics, endothelinconverting enzyme (ECE) inhibitors, endothelin receptor antagonists,natriuretic peptides and their analogs, natriuretic peptide clearancereceptor antagonists, nitric oxide donors, prostaglandin analogs, PDE-Vinhibitors, soluble guanylate cyclase activators and stimulators, andvasopressin receptor antagonists. In one particular embodiment of theinvention, a compound of the invention is combined with an aldosteroneantagonist, a β₁-adrenergic receptor antagonist, an AT₁ receptorantagonist, or a diuretic, and used to treat congestive heart failure.

Diarrhea

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility for the treatment of diarrhea, including infectiousand secretory/watery diarrhea. See, for example, Baumer et al. (1992)Gut 33:753-758; Farthing (2006) Digestive Diseases 24:47-58; andMarcais-Collado (1987) Eur. J. Pharmacol. 144(2):125-132. When used totreat diarrhea, compounds of the invention may be combined with one ormore additional antidiarrheal agents.

Renal Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected toenhance renal function (see Chen et al. (1999) Circulation100:2443-2448; Lipkin et al. (1997) Kidney Int. 52:792-801; and Dussauleet al. (1993) Clin. Sci. 84:31-39) and find utility in treating and/orpreventing renal diseases. Renal diseases of particular interest includediabetic nephropathy, chronic kidney disease, proteinuria, andparticularly acute kidney injury or acute renal failure (see Sharkovskaet al. (2011) Clin. Lab. 57:507-515 and Newaz et al. (2010) RenalFailure 32:384-390). When used to treat renal disease, the compound maybe administered in combination with other therapeutic agents such asangiotensin-converting enzyme inhibitors, AT₁ receptor antagonists, anddiuretics.

Preventative Therapy

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are also expected to be useful in preventative therapy,due to the antihypertrophic and antifibrotic effects of the natriureticpeptides (see Potter et al. (2009) Handbook of Experimental Pharmacology191:341-366), for example in preventing the progression of cardiacinsufficiency after myocardial infarction, preventing arterialrestenosis after angioplasty, preventing thickening of blood vesselwalls after vascular operations, preventing atherosclerosis, andpreventing diabetic angiopathy.

Glaucoma

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are expected to be useful to treat glaucoma. See, forexample, Diestelhorst et al. (1989) International Ophthalmology12:99-101. When used to treat glaucoma, compounds of the invention maybe combined with one or more additional antiglaucoma agents.

Pain Relief

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility as analgesics. See, for example, Rogues et al. (1980)Nature 288:286-288 and Thanawala et al. (2008) Current Drug Targets9:887-894. When used to treat pain, the compounds of the invention maybe combined with one or more additional antinociceptive drugs such asaminopeptidase N or dipeptidyl peptidase III inhibitors, non-steroidalanti-inflammatory agents, monoamine reuptake inhibitors, musclerelaxants, NMDA receptor antagonists, opioid receptor agonists,5-HT_(1D) serotonin receptor agonists, and tricyclic antidepressants.

Other Utilities

Due to their NEP inhibition properties, compounds of the invention arealso expected to be useful as antitussive agents, as well as findutility in the treatment of portal hypertension associated with livercirrhosis (see Sansoe et al. (2005) J. Hepatol. 43:791-798), cancer (seeVesely (2005) J. Investigative Med. 53:360-365), depression (see Nobleet al. (2007) Exp. Opin. Ther. Targets 11:145-159), menstrual disorders,preterm labor, pre-eclampsia, endometriosis, reproductive disorders (forexample, male and female infertility, polycystic ovarian syndrome,implantation failure), and male and female sexual dysfunction, includingmale erectile dysfunction and female sexual arousal disorder. Morespecifically, the compounds of the invention are expected to be usefulin treating female sexual dysfunction (see Pryde et al. (2006) J. Med.Chem. 49:4409-4424), which is often defined as a female patient'sdifficulty or inability to find satisfaction in sexual expression. Thiscovers a variety of diverse female sexual disorders including, by way ofillustration and not limitation, hypoactive sexual desire disorder,sexual arousal disorder, orgasmic disorder and sexual pain disorder.When used to treat such disorders, especially female sexual dysfunction,compounds of the invention may be combined with one or more of thefollowing secondary agents: PDE-V inhibitors, dopamine agonists,estrogen receptor agonists and/or antagonists, androgens, and estrogens.Due to their NEP inhibition properties, compounds of the invention arealso expected to have anti-inflammatory properties, and are expected tohave utility as such, particularly when used in combination withstatins.

Recent studies suggest that NEP plays a role in regulating nervefunction in insulin-deficient diabetes and diet induced obesity. Coppeyet al. (2011) Neuropharmacology 60:259-266. Therefore, due to their NEPinhibition properties, compounds of the invention are also expected tobe useful in providing protection from nerve impairment caused bydiabetes or diet induced obesity.

The amount of the compound of the invention administered per dose or thetotal amount administered per day may be predetermined or it may bedetermined on an individual patient basis by taking into considerationnumerous factors, including the nature and severity of the patient'scondition, the condition being treated, the age, weight, and generalhealth of the patient, the tolerance of the patient to the active agent,the route of administration, pharmacological considerations such as theactivity, efficacy, pharmacokinetics and toxicology profiles of thecompound and any secondary agents being administered, and the like.Treatment of a patient suffering from a disease or medical condition(such as hypertension) can begin with a predetermined dosage or a dosagedetermined by the treating physician, and will continue for a period oftime necessary to prevent, ameliorate, suppress, or alleviate thesymptoms of the disease or medical condition. Patients undergoing suchtreatment will typically be monitored on a routine basis to determinethe effectiveness of therapy. For example, in treating hypertension,blood pressure measurements may be used to determine the effectivenessof treatment. Similar indicators for other diseases and conditionsdescribed herein, are well known and are readily available to thetreating physician. Continuous monitoring by the physician will insurethat the optimal amount of the compound of the invention will beadministered at any given time, as well as facilitating thedetermination of the duration of treatment. This is of particular valuewhen secondary agents are also being administered, as their selection,dosage, and duration of therapy may also require adjustment. In thisway, the treatment regimen and dosing schedule can be adjusted over thecourse of therapy so that the lowest amount of active agent thatexhibits the desired effectiveness is administered and, further, thatadministration is continued only so long as is necessary to successfullytreat the disease or medical condition.

Research Tools

Since compounds of the invention possess NEP enzyme inhibition activity,such compounds are also useful as research tools for investigating orstudying biological systems or samples having a NEP enzyme, for exampleto study diseases where the NEP enzyme or its peptide substrates plays arole. Any suitable biological system or sample having a NEP enzyme maybe employed in such studies which may be conducted either in vitro or invivo. Representative biological systems or samples suitable for suchstudies include, but are not limited to, cells, cellular extracts,plasma membranes, tissue samples, isolated organs, mammals (such asmice, rats, guinea pigs, rabbits, dogs, pigs, humans, and so forth), andthe like, with mammals being of particular interest. In one particularembodiment of the invention, NEP enzyme activity in a mammal isinhibited by administering a NEP-inhibiting amount of a compound of theinvention. Compounds of the invention can also be used as research toolsby conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising aNEP enzyme is typically contacted with a NEP enzyme-inhibiting amount ofa compound of the invention. After the biological system or sample isexposed to the compound, the effects of inhibiting the NEP enzyme aredetermined using conventional procedures and equipment, such as bymeasuring receptor binding in a binding assay or measuringligand-mediated changes in a functional assay. Exposure encompassescontacting cells or tissue with the compound, administering the compoundto a mammal, for example by i.p., p.o, i.v., s.c., or inhaledadministration, and so forth. This determining step can involvemeasuring a response (a quantitative analysis) or can involve making anobservation (a qualitative analysis). Measuring a response involves, forexample, determining the effects of the compound on the biologicalsystem or sample using conventional procedures and equipment, such asenzyme activity assays and measuring enzyme substrate or productmediated changes in functional assays. The assay results can be used todetermine the activity level as well as the amount of compound necessaryto achieve the desired result, that is, a NEP enzyme-inhibiting amount.Typically, the determining step will involve determining the effects ofinhibiting the NEP enzyme.

Additionally, compounds of the invention can be used as research toolsfor evaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds havingNEP-inhibiting activity. In this manner, a compound of the invention isused as a standard in an assay to allow comparison of the resultsobtained with a test compound and with compounds of the invention toidentify those test compounds that have about equal or superioractivity, if any. For example, pK_(i) data for a test compound or agroup of test compounds is compared to the pK_(i) data for a compound ofthe invention to identify those test compounds that have the desiredproperties, for example, test compounds having a pK_(i) value aboutequal or superior to a compound of the invention, if any. This aspect ofthe invention includes, as separate embodiments, both the generation ofcomparison data (using the appropriate assays) and the analysis of testdata to identify test compounds of interest. Thus, a test compound canbe evaluated in a biological assay, by a method comprising the steps of:(a) conducting a biological assay with a test compound to provide afirst assay value; (b) conducting the biological assay with a compoundof the invention to provide a second assay value;

wherein step (a) is conducted either before, after or concurrently withstep (b); and (c) comparing the first assay value from step (a) with thesecond assay value from step (b). Exemplary biological assays include aNEP enzyme inhibition assay.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal),ocular, and parenteral modes of administration. Further, the compoundsof the invention may be administered, for example orally, in multipledoses per day (for example, two, three, or four times daily), in asingle daily dose or a single weekly dose. It will be understood thatany form of the compounds of the invention, (that is, free base, freeacid, pharmaceutically acceptable salt, solvate, etc.) that is suitablefor the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. When discussingcompositions, the “compound of the invention” may also be referred toherein as the “active agent,” to distinguish it from other components ofthe formulation, such as the carrier. Thus, it is understood that theterm “active agent” includes compounds of formula I as well aspharmaceutically acceptable salts, solvates and prodrugs of thatcompound.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,such as in bulk compositions, or less than a therapeutically effectiveamount, that is, individual unit doses designed for multipleadministration to achieve a therapeutically effective amount. Typically,the composition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In oneembodiment, a composition suitable for an oral dosage form, for example,may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. Suitable compositions for oral administration may be inthe form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (capsules,tablets, pills and the like), the composition will typically comprisethe active agent and one or more pharmaceutically acceptable carriers,such as sodium citrate or dicalcium phosphate. Solid dosage forms mayalso comprise: fillers or extenders, such as starches, microcrystallinecellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid;binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants, such as glycerol;disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, optionally with one ormore of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (for example, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.Suspensions may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminium metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, that is, each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, glycerin, and sodium lauryl sulfate. Such compositions aretypically prepared by adding chilled or pressurized hydrofluoroalkane toa suitable container containing the active agent, ethanol (if present)and the surfactant (if present). To prepare a suspension, the activeagent is micronized and then combined with the propellant.Alternatively, a suspension formulation can be prepared by spray dryinga coating of surfactant on micronized particles of the active agent. Theformulation is then loaded into an aerosol canister, which forms aportion of the inhaler.

Compounds of the invention can also be administered parenterally (forexample, by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. Parenteralformulations may also contain one or more anti-oxidants, solubilizers,stabilizers, preservatives, wetting agents, emulsifiers, and dispersingagents. Surfactants, additional stabilizing agents or pH-adjustingagents (acids, bases or buffers) and anti-oxidants are particularlyuseful to provide stability to the formulation, for example, to minimizeor avoid hydrolysis of ester and amide linkages that may be present inthe compound. These formulations may be rendered sterile by use of asterile injectable medium, a sterilizing agent, filtration, irradiation,or heat. In one particular embodiment, the parenteral formulationcomprises an aqueous cyclodextrin solution as the pharmaceuticallyacceptable carrier. Suitable cyclodextrins include cyclic moleculescontaining six or more α-D-glucopyranose units linked at the 1,4positions by a linkages as in amylase, β-cyclodextrin orcycloheptaamylose. Exemplary cyclodextrins include cyclodextrinderivatives such as hydroxypropyl and sulfobutyl ether cyclodextrinssuch as hydroxypropyl-β-cyclodextrin and sulfobutyl ether3-cyclodextrin. Exemplary buffers for such formulations includecarboxylic acid-based buffers such as citrate, lactate and maleatebuffer solutions.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

Secondary Agents

The compounds of the invention may be useful as the sole treatment of adisease or may be combined with one or more additional therapeuticagents in order to obtain the desired therapeutic effect. Thus, in oneembodiment, pharmaceutical compositions of the invention contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”). Such therapeutic agents are wellknown in the art, and include adenosine receptor antagonists,α-adrenergic receptor antagonists, β₁-adrenergic receptor antagonists,β₂-adrenergic receptor agonists, dual-acting β-adrenergic receptorantagonist/α₁-receptor antagonists, advanced glycation end productbreakers, aldosterone antagonists, aldosterone synthase inhibitors,aminopeptidase N inhibitors, androgens, angiotensin-converting enzymeinhibitors and dual-acting angiotensin-converting enzyme/neprilysininhibitors, angiotensin-converting enzyme 2 activators and stimulators,angiotensin-II vaccines, anticoagulants, anti-diabetic agents,antidiarrheal agents, anti-glaucoma agents, anti-lipid agents,antinociceptive agents, anti-thrombotic agents, AT₁ receptor antagonistsand dual-acting AT₁ receptor antagonist/neprilysin inhibitors andmultifunctional angiotensin receptor blockers, bradykinin receptorantagonists, calcium channel blockers, chymase inhibitors, digoxin,diuretics, dopamine agonists, endothelin converting enzyme inhibitors,endothelin receptor antagonists, HMG-CoA reductase inhibitors,estrogens, estrogen receptor agonists and/or antagonists, monoaminereuptake inhibitors, muscle relaxants, natriuretic peptides and theiranalogs, natriuretic peptide clearance receptor antagonists, neprilysininhibitors, nitric oxide donors, non-steroidal anti-inflammatory agents,N-methyl d-aspartate receptor antagonists, opioid receptor agonists,phosphodiesterase inhibitors, prostaglandin analogs, prostaglandinreceptor agonists, renin inhibitors, selective serotonin reuptakeinhibitors, sodium channel blocker, soluble guanylate cyclasestimulators and activators, tricyclic antidepressants, vasopressinreceptor antagonists, and combinations thereof. Specific examples ofthese agents are detailed herein.

Accordingly, in yet another aspect of the invention, a pharmaceuticalcomposition comprises a compound of the invention, a second activeagent, and a pharmaceutically acceptable carrier. Third, fourth etc.active agents may also be included in the composition. In combinationtherapy, the amount of compound of the invention that is administered,as well as the amount of secondary agents, may be less than the amounttypically administered in monotherapy.

Compounds of the invention may be physically mixed with the secondactive agent to form a composition containing both agents; or each agentmay be present in separate and distinct compositions which areadministered to the patient simultaneously or at separate times. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,a compound of the invention can be orally administered simultaneously orsequentially with another active agent using two tablets, with onetablet for each active agent, where sequential may mean beingadministered immediately after administration of the compound of theinvention or at some predetermined time later (for example, one hourlater or three hours later). It is also contemplated that the secondaryagent may be administered more than 24 hours after administration of thecompound of the invention. Alternatively, the combination may beadministered by different routes of administration, that is, one orallyand the other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc.) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount such that they are typically administered in an amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. The secondary agent can be in the formof a pharmaceutically acceptable salt, solvate, optically purestereoisomer, and so forth. The secondary agent may also be in the formof a prodrug, for example, a compound having a carboxylic acid groupthat has been esterified. Thus, secondary agents listed herein areintended to include all such forms, and are commercially available orcan be prepared using conventional procedures and reagents.

In one embodiment, compounds of the invention are administered incombination with an adenosine receptor antagonist, representativeexamples of which include, but are not limited to, naxifylline,rolofylline, SLV-320, theophylline, and tonapofylline.

In one embodiment, compounds of the invention are administered incombination with an α-adrenergic receptor antagonist, representativeexamples of which include, but are not limited to, doxazosin, prazosin,tamsulosin, and terazosin.

Compounds of the invention may also be administered in combination witha β₁-adrenergic receptor antagonist (“β₁-blockers”). Representativeβ₁-blockers include, but are not limited to, acebutolol, alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, bubridine, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol, indenolol, labetolol, levobunolol, mepindolol, metipranolol,metoprolol such as metoprolol succinate and metoprolol tartrate,moprolol, nadolol, nadoxolol, nebivalol, nipradilol, oxprenolol,penbutolol, perbutolol, pindolol, practolol, pronethalol, propranolol,sotalol, sufinalol, talindol, tertatolol, tilisolol, timolol,toliprolol, xibenolol, and combinations thereof. In one particularembodiment, the β₁-antagonist is selected from atenolol, bisoprolol,metoprolol, propranolol, sotalol, and combinations thereof. Typically,the β₁-blocker will be administered in an amount sufficient to providefrom about 2-900 mg per dose.

In one embodiment, compounds of the invention are administered incombination with a β₂-adrenergic receptor agonist, representativeexamples of which include, but are not limited to, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol,salmeterol, terbutaline, vilanterol, and the like Typically, theβ₂-adrenergic receptor agonist will be administered in an amountsufficient to provide from about 0.05-500 mg per dose.

In one embodiment, compounds of the invention are administered incombination with an advanced glycation end product (AGE) breaker,examples of which include, by way of illustration and not limitation,alagebrium (or ALT-711), and TRC4149.

In another embodiment, compounds of the invention are administered incombination with an aldosterone antagonist, representative examples ofwhich include, but are not limited to, eplerenone, spironolactone, andcombinations thereof. Typically, the aldosterone antagonist will beadministered in an amount sufficient to provide from about 5-300 mg perday.

In one embodiment, compounds of the invention are administered incombination with an aminopeptidase N or dipeptidyl peptidase IIIinhibitor, examples of which include, by way of illustration and notlimitation, bestatin and PC18 (2-amino-4-methylsulfonyl butane thiol,methionine thiol).

Compounds of the invention can also be administered in combination withan angiotensin-converting enzyme (ACE) inhibitor. Representative ACEinhibitors include, but are not limited to, accupril, alacepril,benazepril, benazeprilat, captopril, ceranapril, cilazapril, delapril,enalapril, enalaprilat, fosinopril, fosinoprilat, imidapril, lisinopril,moexipril, monopril, moveltipril, pentopril, perindopril, quinapril,quinaprilat, ramipril, ramiprilat, saralasin acetate, spirapril,temocapril, trandolapril, zofenopril, and combinations thereof. In aparticular embodiment, the ACE inhibitor is selected from: benazepril,captopril, enalapril, lisinopril, ramipril, and combinations thereof.Typically, the ACE inhibitor will be administered in an amountsufficient to provide from about 1-150 mg per day.

In another embodiment, compounds of the invention are administered incombination with a dual-acting angiotensin-converting enzyme/neprilysin(ACE/NEP) inhibitor, examples of which include, but are not limited to:AVE-0848((4S,7S,12bR)-7-[3-methyl-2(S)-sulfanylbutyramido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]-benzazepine-4-carboxylicacid); AVE-7688 (ilepatril) and its parent compound; BMS-182657(2-[2-oxo-3 (S)-[3-phenyl-2 (S)-sulfanylpropionamido]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]acetic acid); CGS-35601(N-[1-[4-methyl-2(S)-sulfanylpentanamido]cyclopentylcarbonyl]-L-tryptophan);fasidotril; fasidotrilate; enalaprilat; ER-32935 ((3R,6S,9aR)-6-[3(S)-methyl-2(S)-sulfanylpentanamido]-5-oxoperhydrothiazolo[3,2-a]azepine-3-carboxylicacid); gempatrilat; MDL-101264((4S,7S,12bR)-7-[2(S)-(2-morpholinoacetylthio)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); MDL-101287 ([4S-[4α,7α(R*),12bβ]]-7-[2-(carboxymethyl)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); omapatrilat; RB-105(N-[2(S)-(mercaptomethyl)-3(R)-phenylbutyl]-L-alanine); sampatrilat;SA-898((2R,4R)—N-[2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl)thiazolidin-4-ylcarbonyl]-L-phenylalanine);Sch-50690(N-[1(S)-carboxy-2-[N2-(methanesulfonyl)-L-lysylamino]ethyl]-L-yalyl-L-tyrosine);and combinations thereof, may also be included. In one particularembodiment, the ACE/NEP inhibitor is selected from: AVE-7688,enalaprilat, fasidotril, fasidotrilate, omapatrilat, sampatrilat, andcombinations thereof

In one embodiment, compounds of the invention are administered incombination with an angiotensin-converting enzyme 2 (ACE2) activator orstimulator.

In one embodiment, compounds of the invention are administered incombination with an angiotensin-II vaccine, examples of which include,but are not limited to ATR12181 and CYT006-AngQb.

In one embodiment, compounds of the invention are administered incombination with an anticoagulant, representative examples of whichinclude, but are not limited to: coumarins such as warfarin; heparin;and direct thrombin inhibitors such as argatroban, bivalirudin,dabigatran, and lepirudin.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-diabetic agent. Representative anti-diabeticagents include injectable drugs as well as orally effective drugs, andcombinations thereof. Examples of injectable drugs include, but are notlimited to, insulin and insulin derivatives. Examples of orallyeffective drugs include, but are not limited to: biguanides such asmetformin; glucagon antagonists; α-glucosidase inhibitors such asacarbose and miglitol; dipeptidyl peptidase IV inhibitors (DPP-IVinhibitors) such as alogliptin, denagliptin, linagliptin, saxagliptin,sitagliptin, and vildagliptin; meglitinides such as repaglinide;oxadiazolidinediones; sulfonylureas such as chlorpropamide, glimepiride,glipizide, glyburide, and tolazamide; thiazolidinediones such aspioglitazone and rosiglitazone; and combinations thereof.

In another embodiment, compounds of the invention are administered incombination with antidiarrheal treatments. Representative treatmentoptions include, but are not limited to, oral rehydration solutions(ORS), loperamide, diphenoxylate, and bismuth subsalicylate.

In yet another embodiment, a compound of the invention is administeredin combination with an anti-glaucoma agent. Representative anti-glaucomaagents include, but are not limited to: α-adrenergic agonists such asbrimonidine; β₁-adrenergic receptor antagonists; topical β₁-blockerssuch as betaxolol, levobunolol, and timolol; carbonic anhydraseinhibitors such as acetazolamide, brinzolamide, or dorzolamide;cholinergic agonists such as cevimeline and DMXB-anabaseine; epinephrinecompounds; miotics such as pilocarpine; and prostaglandin analogs.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-lipid agent. Representative anti-lipidagents include, but are not limited to: cholesteryl ester transferprotein inhibitors (CETPs) such as anacetrapib, dalcetrapib, andtorcetrapib; statins such as atorvastatin, fluvastatin, lovastatin,pravastatin, rosuvastatin and simvastatin; and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an anti-thrombotic agent. Representativeanti-thrombotic agents include, but are not limited to: aspirin;anti-platelet agents such as clopidogrel, prasugrel, and ticlopidine;heparin, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an AT₁ receptor antagonist, also known as angiotensinII type 1 receptor blockers (ARBs). Representative ARBs include, but arenot limited to, abitesartan, azilsartan (e.g., azilsartan medoxomil),benzyllosartan, candesartan, candesartan cilexetil, elisartan,embusartan, enoltasosartan, eprosartan, EXP3174, fonsartan, forasartan,glycyllosartan, irbesartan, isoteoline, losartan, medoximil,milfasartan, olmesartan (e.g., olmesartan medoxomil), opomisartan,pratosartan, ripisartan, saprisartan, saralasin, sarmesin, TAK-591,tasosartan, telmisartan, valsartan, zolasartan, and combinationsthereof. In a particular embodiment, the ARB is selected from azilsartanmedoxomil, candesartan cilexetil, eprosartan, irbesartan, losartan,olmesartan medoxomil, saprisartan, tasosartan, telmisartan, valsartan,and combinations thereof. Exemplary salts and/or prodrugs includecandesartan cilexetil, eprosartan mesylate, losartan potassium salt, andolmesartan medoxomil. Typically, the ARB will be administered in anamount sufficient to provide from about 4-600 mg per dose, withexemplary daily dosages ranging from 20-320 mg per day.

Compounds of the invention may also be administered in combination witha dual-acting agent, such as an AT₁ receptor antagonist/neprilysininhibitor (ARB/NEP) inhibitor, examples of which include, but are notlimited to, compounds described in U.S. Publication Nos. 2008/0269305and 2009/0023228, both to Allegretti et al. filed on Apr. 23, 2008, suchas the compound,4′-{2-ethoxy-4-ethyl-5-[(S)-2-mercapto-4-methylpentanoylamino)-methyl]imidazol-1-ylmethyl}-3′-fluorobiphenyl-2-carboxylicacid.

Compounds of the invention may also be administered in combination withmultifunctional angiotensin receptor blockers as described in Kurtz &Klein (2009) Hypertension Research 32:826-834.

In one embodiment, compounds of the invention are administered incombination with a bradykinin receptor antagonist, for example,icatibant (HOE-140). It is expected that this combination therapy maypresent the advantage of preventing angioedema or other unwantedconsequences of elevated bradykinin levels.

In one embodiment, compounds of the invention are administered incombination with a calcium channel blocker. Representative calciumchannel blockers include, but are not limited to, amlodipine, anipamil,aranipine, barnidipine, bencyclane, benidipine, bepridil, clentiazem,cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, etafenone,felodipine, fendiline, flunarizine, gallopamil, isradipine, lacidipine,lercanidipine, lidoflazine, lomerizine, manidipine, mibefradil,nicardipine, nifedipine, niguldipine, niludipine, nilvadipine,nimodipine, nisoldipine, nitrendipine, nivaldipine, perhexiline,prenylamine, ryosidine, semotiadil, terodiline, tiapamil, verapamil, andcombinations thereof. In a particular embodiment, the calcium channelblocker is selected from amlodipine, bepridil, diltiazem, felodipine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, ryosidine, verapamil, andcombinations thereof. Typically, the calcium channel blocker will beadministered in an amount sufficient to provide from about 2-500 mg perdose.

In one embodiment, compounds of the invention are administered incombination with a chymase inhibitor, such as TPC-806 and2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl]acetamide(NK3201).

In one embodiment, compounds of the invention are administered incombination with a diuretic. Representative diuretics include, but arenot limited to: carbonic anhydrase inhibitors such as acetazolamide anddichlorphenamide; loop diuretics, which include sulfonamide derivativessuch as acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone, disulfamide,ethoxzolamide, furosemide, mefruside, methazolamide, piretanide,torsemide, tripamide, and xipamide, as well as non-sulfonamide diureticssuch as ethacrynic acid and other phenoxyacetic acid compounds such astienilic acid, indacrinone and quincarbate; osmotic diuretics such asmannitol; potassium-sparing diuretics, which include aldosteroneantagonists such as spironolactone, and Na⁺ channel inhibitors such asamiloride and triamterene; thiazide and thiazide-like diuretics such asalthiazide, bendroflumethiazide, benzylhydrochlorothiazide,benzthiazide, buthiazide, chlorthalidone, chlorothiazide,cyclopenthiazide, cyclothiazide, epithiazide, ethiazide, fenquizone,flumethiazide, hydrochlorothiazide, hydroflumethiazide, indapamide,methylclothiazide, meticrane, metolazone, paraflutizide, polythiazide,quinethazone, teclothiazide, and trichloromethiazide; and combinationsthereof. In a particular embodiment, the diuretic is selected fromamiloride, bumetanide, chlorothiazide, chlorthalidone, dichlorphenamide,ethacrynic acid, furosemide, hydrochlorothiazide, hydroflumethiazide,indapamide, methylclothiazide, metolazone, torsemide, triamterene, andcombinations thereof. The diuretic will be administered in an amountsufficient to provide from about 5-50 mg per day, more typically 6-25 mgper day, with common dosages being 6.25 mg, 12.5 mg or 25 mg per day.

Compounds of the invention may also be administered in combination withan endothelin converting enzyme (ECE) inhibitor, examples of whichinclude, but are not limited to, phosphoramidon, CGS 26303, andcombinations thereof

In a particular embodiment, compounds of the invention are administeredin combination with an endothelin receptor antagonist. Representativeendothelin receptor antagonists include, but are not limited to:selective endothelin receptor antagonists that affect endothelin Areceptors, such as avosentan, ambrisentan, atrasentan, BQ-123,clazosentan, darusentan, sitaxentan, and zibotentan; and dual endothelinreceptor antagonists that affect both endothelin A and B receptors, suchas bosentan, macitentan, tezosentan).

In yet another embodiment, a compound of the invention is administeredin combination with one or more HMG-CoA reductase inhibitors, which arealso known as statins. Representative statins include, but are notlimited to, atorvastatin, fluvastatin, lovastatin, pitavastatin,pravastatin, rosuvastatin and simvastatin.

In one embodiment, compounds of the invention are administered incombination with a monoamine reuptake inhibitor, examples of whichinclude, by way of illustration and not limitation, norepinephrinereuptake inhibitors such as atomoxetine, buproprion and the buproprionmetabolite hydroxybuproprion, maprotiline, reboxetine, and viloxazine;selective serotonin reuptake inhibitors (SSRIs) such as citalopram andthe citalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline;dual serotonin-norepinephrine reuptake inhibitors (SNRIs) such asbicifadine, duloxetine, milnacipran, nefazodone, and venlafaxine; andcombinations thereof

In another embodiment, compounds of the invention are administered incombination with a muscle relaxant, examples of which include, but arenot limited to: carisoprodol, chlorzoxazone, cyclobenzaprine,diflunisal, metaxalone, methocarbamol, and combinations thereof

In one embodiment, compounds of the invention are administered incombination with a natriuretic peptide or analog, examples of whichinclude but are not limited to: carperitide, CD-NP (Nile Therapeutics),CU-NP, nesiritide, PL-3994 (Palatin Technologies, Inc.), ularitide,cenderitide, and compounds described in Ogawa et al (2004) J. Biol.Chem. 279:28625-31. These compounds are also referred to as natriureticpeptide receptor-A (NPR-A) agonists. In another embodiment, compounds ofthe invention are administered in combination with a natriuretic peptideclearance receptor (NPR-C) antagonist such as SC-46542, cANF (4-23), andAP-811 (Veale (2000) Bioorg Med Chem Lett 10:1949-52). For example,AP-811 has shown synergy when combined with the NEP inhibitor, thiorphan(Wegner (1995) Clin. Exper. Hypert. 17:861-876).

In another embodiment, compounds of the invention are administered incombination with a neprilysin (NEP) inhibitor. Representative NEPinhibitors include, but are not limited to: AHU-377; candoxatril;candoxatrilat; dexecadotril((+)-N-[2(R)-(acetylthiomethyl)-3-phenylpropionyl]glycine benzyl ester);CGS-24128(3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-24592((S)-3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-25155 (N-[9(R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl]-4(R)-hydroxy-L-prolinebenzyl ester); 3-(1-carbamoylcyclohexyl)propionic acid derivativesdescribed in WO 2006/027680 to Hepworth et al. (Pfizer Inc.); JMV-390-1(2(R)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine);ecadotril; phosphoramidon; retrothiorphan; RU-42827(2-(mercaptomethyl)-N-(4-pyridinyl)benzenepropionamide); RU-44004(N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl)propionamide); SCH-32615((S)—N—[N-(1-carboxy-2-phenylethyl)-L-phenylalanyl]-β-alanine) and itsprodrug SCH-34826((S)—N—[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine);sialorphin; SCH-42495(N-[2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl)propionyl]-L-methionineethyl ester); spinorphin; SQ-28132(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]leucine); SQ-28603(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-β-alanine); SQ-29072(7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid);thiorphan and its prodrug racecadotril; UK-69578(cis-4-[[[1-[2-carboxy-3-(2-methoxyethoxy)propyl]cyclopentyl]carbonyl]amino]cyclohexanecarboxylicacid); UK-447,841(2-{1-[3-(4-chlorophenyl)propylcarbamoyl]-cyclopentylmethyl}-4-methoxybutyricacid); UK-505,749((R)-2-methyl-3-{1-[3-(2-methylbenzothiazol-6-yl)propylcarbamoyl]cyclopentyl}propionicacid); 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid and 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid ethyl ester (WO 2007/056546); daglutril[(3S,2′R)-3-{1-[2′-(ethoxycarbonyl)-4′-phenylbutyl]-cyclopentan-1-carbonylamino}-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-aceticacid] described in WO 2007/106708 to Khder et al. (Novartis AG); andcombinations thereof. In a particular embodiment, the NEP inhibitor isselected from AHU-377, candoxatril, candoxatrilat, CGS-24128,phosphoramidon, SCH-32615, SCH-34826, SQ-28603, thiorphan, andcombinations thereof. In a particular embodiment, the NEP inhibitor is acompound such as daglutril or CGS-26303([N-[2-(biphenyl-4-yl)-1(S)-(1H-tetrazol-5-yl)ethyl]amino]methylphosphonicacid), which have activity both as inhibitors of the endothelinconverting enzyme (ECE) and of NEP. Other dual acting ECE/NEP compoundscan also be used. The NEP inhibitor will be administered in an amountsufficient to provide from about 20-800 mg per day, with typical dailydosages ranging from 50-700 mg per day, more commonly 100-600 or 100-300mg per day.

In one embodiment, compounds of the invention are administered incombination with a nitric oxide donor, examples of which include, butare not limited to nicorandil; organic nitrates such as pentaerythritoltetranitrate; and sydnonimines such as linsidomine and molsidomine.

In yet another embodiment, compounds of the invention are administeredin combination with a non-steroidal anti-inflammatory agent (NSAID).Representative NSAIDs include, but are not limited to: acemetacin,acetyl salicylic acid, alclofenac, alminoprofen, amfenac, amiprilose,aloxiprin, anirolac, apazone, azapropazone, benorilate, benoxaprofen,bezpiperylon, broperamole, bucloxic acid, carprofen, clidanac,diclofenac, diflunisal, diftalone, enolicam, etodolac, etoricoxib,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, fluprofen, flurbiprofen, furofenac,ibufenac, ibuprofen, indomethacin, indoprofen, isoxepac, isoxicam,ketoprofen, ketorolac, lofemizole, lornoxicam, meclofenamate,meclofenamic acid, mefenamic acid, meloxicam, mesalamine, miroprofen,mofebutazone, nabumetone, naproxen, niflumic acid, oxaprozin, oxpinac,oxyphenbutazone, phenylbutazone, piroxicam, pirprofen, pranoprofen,salsalate, sudoxicam, sulfasalazine, sulindac, suprofen,tenoxicam,tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid, tolmetin,triflumidate, zidometacin, zomepirac, and combinations thereof. In aparticular embodiment, the NSAID is selected from etodolac,flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meloxicam,naproxen, oxaprozin, piroxicam, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an N-methyl d-aspartate (NMDA) receptor antagonist,examples of which include, by way of illustration and not limitation,including amantadine, dextromethorphan, dextropropoxyphene, ketamine,ketobemidone, memantine, methadone, and so forth.

In still another embodiment, compounds of the invention are administeredin combination with an opioid receptor agonist (also referred to asopioid analgesics). Representative opioid receptor agonists include, butare not limited to: buprenorphine, butorphanol, codeine, dihydrocodeine,fentanyl, hydrocodone, hydromorphone, levallorphan, levorphanol,meperidine, methadone, morphine, nalbuphine, nalmefene, nalorphine,naloxone, naltrexone, nalorphine, oxycodone, oxymorphone, pentazocine,propoxyphene, tramadol, and combinations thereof. In certainembodiments, the opioid receptor agonist is selected from codeine,dihydrocodeine, hydrocodone, hydromorphone, morphine, oxycodone,oxymorphone, tramadol, and combinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with a phosphodiesterase (PDE) inhibitor, particularly aPDE-V inhibitor. Representative PDE-V inhibitors include, but are notlimited to, avanafil, lodenafil, mirodenafil, sildenafil (Revatio®),tadalafil (Adcirca®), vardenafil (Levitra®), and udenafil.

In another embodiment, compounds of the invention are administered incombination with a prostaglandin analog (also referred to as prostanoidsor prostacyclin analogs). Representative prostaglandin analogs include,but are not limited to, beraprost sodium, bimatoprost, epoprostenol,iloprost, latanoprost, tafluprost, travoprost, and treprostinil, withbimatoprost, latanoprost, and tafluprost being of particular interest.

In yet another embodiment, compounds of the invention are administeredin combination with a prostaglandin receptor agonist, examples of whichinclude, but are not limited to, bimatoprost, latanoprost, travoprost,and so forth.

Compounds of the invention may also be administered in combination witha renin inhibitor, examples of which include, but are not limited to,aliskiren, enalkiren, remikiren, and combinations thereof

In another embodiment, compounds of the invention are administered incombination with a selective serotonin reuptake inhibitor (SSRI).Representative SSRIs include, but are not limited to: citalopram and thecitalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a 5-HT_(1D) serotonin receptor agonist, examples ofwhich include, by way of illustration and not limitation, triptans suchas almotriptan, avitriptan, eletriptan, frovatriptan, naratriptanrizatriptan, sumatriptan, and zolmitriptan.

In one embodiment, compounds of the invention are administered incombination with a sodium channel blocker, examples of which include, byway of illustration and not limitation, carbamazepine, fosphenytoin,lamotrigine, lidocaine, mexiletine, oxcarbazepine, phenytoin, andcombinations thereof.

In one embodiment, compounds of the invention are administered incombination with a soluble guanylate cyclase stimulator or activator,examples of which include, but are not limited to ataciguat, riociguat,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a tricyclic antidepressant (TCA), examples of whichinclude, by way of illustration and not limitation, amitriptyline,amitriptylinoxide, butriptyline, clomipramine, demexiptiline,desipramine, dibenzepin, dimetacrine, dosulepin, doxepin, imipramine,imipraminoxide, lofepramine, melitracen, metapramine, nitroxazepine,nortriptyline, noxiptiline, pipofezine, propizepine, protriptyline,quinupramine, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a vasopressin receptor antagonist, examples of whichinclude, by way of illustration and not limitation, conivaptan andtolvaptan.

Combined secondary therapeutic agents may also be helpful in furthercombination therapy with compounds of the invention. For example,compounds of the invention can be combined with a diuretic and an ARB,or a calcium channel blocker and an ARB, or a diuretic and an ACEinhibitor, or a calcium channel blocker and a statin. Specific examplesinclude, a combination of the ACE inhibitor enalapril (in the maleatesalt form) and the diuretic hydrochlorothiazide, which is sold under themark Vaseretic®, or a combination of the calcium channel blockeramlodipine (in the besylate salt form) and the ARB olmesartan (in themedoxomil prodrug form), or a combination of a calcium channel blockerand a statin, all may also be used with the compounds of the invention.Other therapeutic agents such as α₂-adrenergic receptor agonists andvasopressin receptor antagonists may also be helpful in combinationtherapy. Exemplary α₂-adrenergic receptor agonists include clonidine,dexmedetomidine, and guanfacine.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), 440 g spray-dried lactose and 10 gmagnesium stearate are thoroughly blended. The resulting composition isthen loaded into hard gelatin capsules (500 mg of composition percapsule). Alternately, a compound of the invention (20 mg) is thoroughlyblended with starch (89 mg), microcrystalline cellulose (89 mg) andmagnesium stearate (2 mg). The mixture is then passed through a No. 45mesh U.S. sieve and loaded into a hard gelatin capsule (200 mg ofcomposition per capsule).

Alternately, a compound of the invention (30 g), a secondary agent (20g), 440 g spray-dried lactose and 10 g magnesium stearate are thoroughlyblended, and processed as described above.

Exemplary Gelatin Capsule Formulation for Oral Administration

A compound of the invention (100 mg) is thoroughly blended withpolyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg).The mixture is then loaded into a gelatin capsule (400 mg of compositionper capsule). Alternately, a compound of the invention (70 mg) and asecondary agent (30 mg) are thoroughly blended with polyoxyethylenesorbitan monooleate (50 mg) and starch powder (250 mg), and theresulting mixture loaded into a gelatin capsule (400 mg of compositionper capsule).

Alternately, a compound of the invention (40 mg) is thoroughly blendedwith microcrystalline cellulose (Avicel PH 103; 259.2 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

A compound of the invention (10 mg), starch (45 mg) and microcrystallinecellulose (35 mg) are passed through a No. 20 mesh U.S. sieve and mixedthoroughly. The granules so produced are dried at 50-60° C. and passedthrough a No. 16 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4mg as a 10% solution in sterile water) is mixed with sodiumcarboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg), and talc (1mg), and this mixture is then passed through a No. 16 mesh U.S. sieve.The sodium carboxymethyl starch, magnesium stearate and talc are thenadded to the granules. After mixing, the mixture is compressed on atablet machine to afford a tablet weighing 100 mg.

Alternately, a compound of the invention (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, a compound of the invention (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of composition per tablet).

Alternately, a compound of the invention (100 mg) is thoroughly blendedwith cornstarch (100 mg) with an aqueous solution of gelatin (20 mg).The mixture is dried and ground to a fine powder. Microcrystallinecellulose (50 mg) and magnesium stearate (5 mg) are then admixed withthe gelatin formulation, granulated and the resulting mixture compressedto form tablets (100 mg of the compound of the invention per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of the compound of the invention per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K(magnesium aluminum silicate) 1.0 g Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Liquid Formulation for Oral Administration

A suitable liquid formulation is one with a carboxylic acid-based buffersuch as citrate, lactate and maleate buffer solutions. For example, acompound of the invention (which may be pre-mixed with DMSO) is blendedwith a 100 mM ammonium citrate buffer and the pH adjusted to pH 5, or isblended with a 100 mM citric acid solution and the pH adjusted to pH 2.Such solutions may also include a solubilizing excipient such as acyclodextrin, for example the solution may include 10 wt %hydroxypropyl-β-cyclodextrin.

Other suitable formulations include a 5% NaHCO₃ solution, with orwithout cyclodextrin.

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, a compound of the invention (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1 NNaOH. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard, generally accepted meaning:

AcOH acetic acid BOC t-butoxycarbonyl (—C(O)OC(CH₃)₃) (BOC)₂O di-t-butyldicarbonate Bn benzyl DCC dicyclohexylcarbodiimide DCM dichloromethaneor methylene chloride DIBAL diisobutylaluminum hydride DIPEAN,N-diisopropylethylamine DMAP 4-dimethylaminopyridine DMFN,N-dimethylformamide DMSO dimethyl sulfoxide Dnp 2,4-dinitrophenyl EDC1-(3-dimethylaminopropyl)-3- ethylcarbodiimide Et₃N triethylamine Et₂Odiethyl ether EtOAc ethyl acetate EtOH ethanol HEPES4-(2-hydroxyethyl)-1-piperazineethane- sulfonic acid HOBt1-hydroxybenzotriazole LiHMDS lithium hexamethyl disilazide Mca(7-methoxycoumarin-4-yl)acyl MeCN acetonitrile MeOH methanol MTBE methylt-butyl ether NaHMDS sodium hexamethyldisilazide Pd(dppf)₂Cl₂1,1-bis(diphenylphosphino) ferrocene palladium chloride Pd(PPh₃)₄tetrakis(triphenylphosphine)palladium(0) PE petroleum etherSilicaCat ® DPP-Pd silica based diphenylphosphine palladium (II)catalyst SilicaCat ® Pd(0) silica based palladium (0) catalyst TFAtrifluoroacetic acid THF tetrahydrofuran

Unless noted otherwise, all materials, such as reagents, startingmaterials and solvents, were purchased from commercial suppliers (suchas Sigma-Aldrich, Fluka Riedel-de Haen, and the like) and were usedwithout further purification.

Reactions were run under nitrogen atmosphere, unless noted otherwise.The progress of reactions were monitored by thin layer chromatography(TLC), analytical high performance liquid chromatography (anal. HPLC),and mass spectrometry, the details of which are given in specificexamples. Solvents used in analytical HPLC were as follows: solvent Awas 98% H₂O/2% MeCN/1.0 mL/L TFA; solvent B was 90% MeCN/10% H₂O/1.0mL/L TFA.

Reactions were worked up as described specifically in each preparationfor example; commonly reaction mixtures were purified by extraction andother purification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC, typically using Microsorb C18and Microsorb BDS column packings and conventional eluents. Progress ofreactions was typically measured by liquid chromatography massspectrometry (LCMS). Characterization of isomers were done by NuclearOverhauser effect spectroscopy (NOE). Characterization of reactionproducts was routinely carried out by mass and ¹H-NMR spectrometry. ForNMR measurement, samples were dissolved in deuterated solvent (CD₃OD,CDCl₃, or DMSO-d₆), and ¹H-NMR spectra were acquired with a VarianGemini 2000 instrument (400 MHz) under standard observation conditions.Mass spectrometric identification of compounds was typically conductedusing an electrospray ionization method (ESMS) with an AppliedBiosystems (Foster City, Calif.) model API 150 EX instrument or anAgilent (Palo Alto, Calif.) model 1200 LC/MSD instrument.

Preparation 1 Acetoxy(diethoxyphosphoryl)acetic Acid Ethyl Ester

Ethyl 2-oxoacetate (50%) (74 g, 724.8 mmol) was added dropwise withstirring at 0° C. to a solution of diethyl hydrogen phosphite (50 g,362.1 mmol) in toluene (100 mL), under nitrogen. Et₃N (110 g, 1.1 mol)was added dropwise with stirring at 0° C. The resulting solution wasstirred for 1 hour at room temperature. To the mixture was added aceticanhydride (37 g, 362.4 mmol) dropwise with stirring at 0° C. Theresulting solution was stirred overnight at room temperature. The pHvalue of the solution was adjusted to 6 with 2N HCl. The resultingsolution was extracted with DCM (3×150 mL) and the organic layers werecombined, dried over Na₂SO₄, and concentrated under vacuum. The residuewas loaded onto a silica gel column with EtOAc:hexanes (1:2˜1:5) toyield the title compound (52 g) as a light yellow liquid.

Preparation 2 (R)-4-Amino-5-biphenyl-4-yl-2-hydroxypentanoic Acid EthylEster

A solution of acetoxy(diethoxyphosphoryl)acetic acid ethyl ester (15.6g, 55.3 mmol, 1.2 equiv) in THF (dried) (150 mL), under nitrogen, wascooled to −78° C. LiHMDS (1M in THF) (55.3 mL) was added dropwise withstirring at −78° C. After stirring for 30 minutes at that temperature, asolution of crude ((R)-2-biphenyl-4-yl-1-formylethyl)carbamic acidt-butyl ester (15.0 g, 1.0 eq.) in THF (dried) (30 mL) was addeddropwise over 15 minutes. Stirring was continued for 1.5 hours at −78°C. before the mixture was poured into a cold solution with water (200mL) and EtOAc (200 mL). The organic layer was repeatedly separated andthe aqueous layer was re-extracted with EtOAc (2×100 mL). The combinedorganic layers were dried over Na₂SO₄, filtered, and evaporated, and theresidue was purified by flash chromatography

(EtOAc/hexanes=0˜1:10) to give Compound 1 (10.5 g) as a white solid.

A stirred solution of Compound 1 (10.5 g, 23.2 mmol) in EtOH (anhydrous)(100 mL) was combined with palladium carbon (1.0 g), under nitrogen. Themixture was purged four times with hydrogen and then hydrogen wasbubbled over 2 hours at room temperature. The palladium carbon wasfiltered out, and the filtrate was concentrated under vacuum to yieldcrude Compound 2 (10.0 g) as a pale-yellow oil, which was used withoutfurther purification.

Compound 2 (10.0 g, 22.0 mmol) in EtOH (anhydrous) (100 mL) was combinedwith potassium carbonate (6.1 g, 44.1 mmol) and the resulting solutionwas stirred for 2 hours at room temperature. The solids were filteredout and the filtrate was concentrated under vacuum. The residue wasloaded onto a silica gel column (EtOAc/hexanes=0˜1:5) to yield Compound3 (6.0 g) as a white solid.

Compound 3 (6.0 g, 14.5 mmol) was dissolved in DCM (dried) (120 mL), andHCl was bubbled into the mixture over 5˜6 hours at room temperature.Solid precipitate was observed. The mixture was concentrated to halfvolume then filtered. The solids were collected and washed with coldEtOAc, and dried over reduced pressure to yield the title compound (4.2g) as an off-white solid HCl salt. LC-MS (ES, m/z): 314 [M−HCl+H]⁺.

¹H NMR (300 MHz, DMSO): δ (ppm)=8.07 (s, 1.9H), 7.96 (s, 1.2H),7.65-7.69 (m, 4.0H), 7.45-7.5.0 (m, 2.0H), 7.33-7.39 (m, 3.0H),6.05-6.07 (m, 0.63H), 5.88-5.90 (m, 0.88H), 4.32-4.38 (m, 0.80H),4.18-4.31 (m, 0.51H), 4.05-4.11 (m, 2H), 3.50 (s, 1H), 2.75-3.05 (m,2.8H), 1.83-1.94 (m, 1H), 1.71-1.82 (m, 1H), 1.10-1.20 (m, 3.3H).

Preparation 3 (S)-2-(4-Bromobenzyl)-5-oxopyrrolidine-1-carboxylic Acidt-Butyl Ester

To a solution of (R)-2-amino-3-(4-bromophenyl)propionic acid (50 g, 0.2mol) in MeCN (700 mL) was added a solution of NaOH (16.4 g, 0.4 mol) inwater (700 mL) at −5° C. After stirring for 10 minutes, a solution of(BOC)₂O (44.7 g, 0.2 mol) in MeCN (100 mL) was added. The mixture waswarmed to room temperature and stirred overnight. After the evaporationof the MeCN, the residue was diluted with DCM (800 mL) and acidifiedwith 1 M HCl to pH 2 at −5° C. The aqueous was extracted with DCM (3×200mL). The combined organic layers were washed with saturated aqueous NaCl(500 mL), dried over Na₂SO₄ and concentrated to yield Compound 1 (66.5g) as a white solid. LC-MS: 366 (M+Na), 709 (2M+Na).

To a solution of Compound 1 (66.5 g, 193 μmol), Meldrum's acid (33.4 g,232 mmol) and DMAP (37.7 g, 309 mmol) in anhydrous DCM (600 mL), wasadded dropwise a solution of DCC (47.9 g, 232 mmol) in anhydrous DCM(200 mL) over 1 hour at −5° C. under nitrogen. The mixture was stirredat −5° C. for 8 hours, then refrigerated overnight. Crystals ofdicyclohexylurea were observed. The mixture was filtered, washed with 5%KHSO₄ (5×200 mL) and saturated aqueous NaCl (200 mL), then dried overanhydrous MgSO₄ under refrigeration overnight. The solution was thenevaporated to yield crude Compound 2 (91 g) as a light yellow solid.LC-MS: 492(M+Na), 961(2M+Na).

To a solution of crude Compound 2 (91 g, 193 mmol) in anhydrous DCM (1L) was added AcOH (127.5 g, 2.1 mol) at −5° C. under nitrogen. Themixture was stirred at −5° C. for 30 minutes, then NaBH₄ (18.3 g, 483mmol) was added in small portions over 1 hour. After stirring foranother 1 hour at −5° C., saturated aqueous NaCl (500 mL) was added. Theorganic layer was washed with saturated aqueous NaCl (2×300 mL) andwater (2×300 mL), dried over MgSO₄, filtered, and concentrated to yieldthe crude product, which was further purified by washing with Et₂O toyield Compound 3 (68 g) as a light yellow solid. LC-MS: 478 (M+Na), 933(2M+Na).

A solution of Compound 3 (68 g, 149 mmol) in anhydrous toluene (500 mL)was refluxed under nitrogen for 3 hours. After evaporation of thesolvent, the residue was purified by chromatography (hexanes:EtOAc=10:1)to yield the title compound (38 g) as a light yellow oil. LC-MS: 376(M+Na), 729 (2M+Na).

Preparation 4 (2R,4R)-4-Amino-5-(4-bromophenyl)-2-hydroxypentanoic AcidEthyl Ester

To a solution of (S)-2-(4-bromobenzyl)-5-oxopyrrolidine-1-carboxylicacid t-butyl ester (38 g, 107 mmol) in anhydrous DCM (250 mL) was addedTFA (20 mL, 0.27 mol) at −5° C. under nitrogen. The mixture was warmedto room temperature and stirred overnight. After evaporation of thesolvent, the residue was diluted with EtOAc (300 mL) and washed withsaturated aqueous NaHCO₃ (3×200 mL), water (200 mL), saturated aqueousNaCl (250 mL), dried over Na₂SO₄ and concentrated to yield crudeCompound 1 (24 g) as a light yellow solid. LC-MS: 254 [M+H].

To a solution of NaH (8.6 g, 250 mmol) in anhydrous THF (200 mL) wasadded dropwise a solution of Compound 1 (24 g, 94 mmol) in anhydrous THF(200 mL) over 30 minutes at 0° C. under nitrogen. The mixture was warmedto room temperature and stirred for 2 hours. After cooling to 0° C.,pivaloyl chloride (18 g, 150 mmol) was added dropwise over 30 minutes.The mixture was warmed to room temperature and stirred overnight. Thereaction was quenched with saturated aqueous NH₄Cl (300 mL) andextracted with EtOAc (3×200 mL). The combined organic layers were washedwith saturated aqueous NaCl (300 mL), dried over MgSO₄, filtered andconcentrated to yield the crude product, which was further purified bychromatography (hexanes:EtOAc=25:1) to yield Compound 2 (18 g) as alight yellow solid. LC-MS: 360 (M+Na).

To a solution of Compound 2 (18 g, 53 mmol) in anhydrous THF (250 mL)was added dropwise NaHMDS (47.7 mL, 96 mmol) over 30 minutes at −78° C.under nitrogen. After stirring at −78° C. for 90 minutes, a solution of(+)-(8,8-dichlorocamphorylsulfonyl)-oxaziridine (31.6 g, 106 mmol) wasadded dropwise over 30 minutes. After stirring at −78° C. for 2 hours,the reaction was quenched with saturated aqueous NH₄Cl (400 mL) andextracted with EtOAc (3×300 mL). The combined organic layers were washedwith saturated aqueous NaCl (300 mL), dried over MgSO₄, filtered, andconcentrated to give the crude product which was further purified bychromatography (hexanes:EtOAc=15:1) to yield Compound 3 (8.9 g) as alight yellow solid. LC-MS: 376 (M+Na).

A solution of Compound 3 (8.9 g, 25 mmol) in concentrated HCl (81 mL, 81mmol) was heated at 100° C. for 16 hours. The mixture was thenconcentrated to yield the crude product which was further purified bywashing with Et₂O to yield compound 4 (7 g) as a light yellow solid HClsalt. LC-MS: 323 (M+H).

A solution of compound 4 (7 g, 22 mmol) in EtOH (10 mL) was combinedwith 8M HCl in EtOH (120 mL, 960 mmol) at room temperature. The mixturewas heated at 50° C. for 16 hours, then concentrated. The crude productwas further purified by washing with Et₂O to yield the title compound (6g) as a light yellow solid HCl salt. LC-MS: 352 (M+H).

Preparation 5(3R,5R)-5-(3′-Chlorobiphenyl-4-ylmethyl)-1-(2,2-dimethylpropionyl)-3-hydroxypyrrolidin-2-one

To a solution of (S)-2-(4-bromobenzyl)-5-oxopyrrolidine-1-carboxylicacid t-butyl ester (15 g, 43 mmol) in 1,4-dioxane (600 mL) was added3-chlorophenylboronic acid (8 g, 51 mmol) and Pd(dppf)₂Cl₂ (3.1 g, 4.2mmol) at room temperature under nitrogen. After stirring for 10 minutes,a solution of K₂CO₃ (11.7 g, 85 mmol) in water (60 mL) was added. Themixture was heated to 60° C. and stirred overnight. After evaporation ofthe solvent, water (200 mL) was added and extracted with EtOAc (3×200mL). The combined organic layers were washed with saturated aqueous NaCl(400 mL), dried over Na₂SO₄, and concentrated to yield the crude productwhich was further purified by column chromatography (hexanes:EtOAc=6:1)to yield Compound 1 (15 g) as a light yellow solid. LC-MS: 408 (M+Na).

To a solution of Compound 1 (15 g, 0.039 mol) in anhydrous DCM (250 mL)was added TFA (20 mL, 270 mmol) at −5° C. under nitrogen. The mixturewas warmed to room temperature and stirred overnight. After evaporationof the solvent, the residue was diluted with EtOAc (300 mL), then washedwith saturated aqueous NaHCO₃ (3×200 mL), water (200 mL), and saturatedaqueous NaCl (250 mL), then dried over Na₂SO₄ and concentrated to yieldcrude Compound 2 (11 g) as a light yellow solid. LC-MS: 286 [M+H].

To a solution of NaH (2.3 g, 98 mmol) in anhydrous THF (200 mL) wasadded dropwise a solution of Compound 2 (11 g, 39 mmol) in anhydrous THF(100 mL) over 30 minutes at 0° C. under nitrogen. The mixture was warmedto room temperature and stirred for 2 hours. After cooling to 0° C.,pivaloyl chloride (6 g, 51 mmol) was added dropwise over 30 minutes. Themixture was warmed to room temperature and stirred overnight. Thereaction was quenched with saturated aqueous NH₄Cl (200 mL) andextracted with EtOAc (3×200 mL). The combined organic layers were washedwith saturated aqueous NaCl (300 mL), dried over MgSO₄, filtered, andconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=25:1) to yield Compound 3 (10.5 g) as alight yellow solid. LC-MS: 391 (M+Na).

To a solution of Compound 3 (10.5 g, 29 mmol) in anhydrous THF (120 mL)was added dropwise NaHMDS (29 mL, 58 mmol) over 30 minutes at −78° C.under nitrogen. After stirring at −78° C. for 90 minutes, a solution of(+)-(8,8-dichlorocamphorylsulfonyl)-oxaziridine (15.6 g, 52 mmol) wasadded dropwise over 30 minutes. After stirring at −78° C. for 2 hours,the reaction was quenched with saturated NH₄Cl (400 mL) and extractedwith EtOAc (3×300 mL). The combined organic layers were washed withsaturated aqueous NaCl (300 mL), dried over MgSO₄, filtered, andconcentrated to give the crude product which was further purified bychromatography (hexanes:EtOAc=15:1) to yield the title compound (9.6 g)as a light yellow solid. LC-MS: 408 (M+Na).

Preparation 6(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic Acid EthylEster

A solution of(3R,5R)-5-(3′-chlorobiphenyl-4-ylmethyl)-1-(2,2-dimethylpropionyl)-3-hydroxypyrrolidin-2-one(9.6 g, 25 mmol) in concentrated HCl (81 mL, 81 mmol) was heated at 100°C. for 16 hours. The mixture was then concentrated to give the crudeproduct which was further purified by washing with Et₂O to yieldCompound 1 (5.7 g) as a light yellow solid HCl salt. LC-MS: 320 (M+H).

To a solution of Compound 1 (5.7 g, 18 mmol) in EtOH (10 mL) was added8M HCl in EtOH (120 mL, 960 mmol) at room temperature. The mixture washeated at 50° C. for 16 hours. After concentration, the crude productwas further purified by washing with Et₂O to yield the title compound(2.1 g) as a light yellow solid HCl salt. LC-MS: 348 (M+H).

Preparation 7(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic Acid

1 M aqueous HCl (2.0 mmol) was added to(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (150.0 mg, 431 μmol) and the mixture was stirred at 100° C. for 2hours. The mixture was concentrated under vacuum for 3 hours and theresidue was purified by reverse phase to yield the title compound (117mg) as a white solid.

Preparation 8 Chloro-Oxo-Acetic Acid Isopropyl Ester

Isopropanol (158 μL, 2.1 mmol, 1.0 eq.) was added dropwise over 5minutes to oxalyl chloride (350 μL, 4.14 mmol, 2.0 eq.) at 0° C., andthe resulting mixture was stirred at room temperature for 2 hours. Theexcess oxalyl chloride was removed by rotary evaporation (40° C., 50mmHg) and used without further purification.

Preparation 9 Chloro-Oxo-Acetic Acid Isobutyl Ester

Isobutanol (191 μL, 2.1 mmol, 1.0 eq.) was added dropwise over 5 minutesto oxalyl chloride (350 μL, 4.14 mmol, 2.0 eq.) at 0° C., and theresulting mixture was stirred at room temperature for 2 hours. Theexcess oxalyl chloride was removed by rotary evaporation (40° C., 40mmHg) and used without further purification.

Preparation 10 t-Butyl Oxalyl Chloride

Oxalyl chloride (274 μL, 3.2 mmol) was added to a solution of t-butylalcohol (289 μL, 3.0 mmol) in ether (2.0 mL, 19.0 mmol) and the mixturewas stirred at room temperature for 1 hour and then concentrated invacuo to yield a clear colorless liquid. An approximately 1M solution oft-butyl oxalyl chloride was prepared by dissolving the resulting clearcolorless liquid in DCM (˜3.0 mL).

Preparation 11 Chloro-Oxo-Acetic Acid 2-Methoxyethyl Ester

A solution of 2-methoxyethanol (295 mg, 3.9 mmol) in DCM (total volume:0.5 mL) was added to a solution of oxalyl chloride (0.5 mL, 5.8 mmol) inDCM (total volume 1.0 mL) at 0° C. and the resulting mixture was stirredat room temperature for 30 minutes. The mixture was concentrated invacuo and the resulting residue was dissolved in DCM (3.9 mL) to yield a1.0M solution in DCM.

Preparation 12 Chloro-Oxo-Acetic Acid 3-Ethoxypropyl Ester

A solution of 3-ethoxypropan-1-ol (404 mg, 3.9 mmol) in DCM (totalvolume: 0.5 mL) was added to a solution of oxalyl chloride (0.5 mL, 5.8mmol) in DCM (total volume 1.0 mL) at 0° C. and the resulting mixturewas stirred at room temperature for 30 minutes. The mixture wasconcentrated in vacuo and the resulting residue was dissolved in DCM(3.9 mL) to yield a 1.0M solution in DCM.

Preparation 13 Chloro-Oxo-Acetic Acid 2-Phenoxyethyl Ester

A solution of 2-phenoxyethanol (536 mg, 3.9 mmol) in DCM (total volume:0.5 mL) was added to a solution of oxalyl chloride (0.5 mL, 5.8 mmol) inDCM (total volume 1.0 mL) at 0° C. and the resulting mixture was stirredat room temperature for 30 minutes. The mixture was concentrated invacuo and the resulting residue was dissolved in DCM (3.9 mL) to yield a1.0M solution in DCM.

Preparation 14(2R,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicAcid

A solution of(3R,5R)-5-(3′-chlorobiphenyl-4-ylmethyl)-1-(2,2-dimethylpropionyl)-3-hydroxypyrrolidin-2-one(4.5 g, 11.7 mmol) in concentrated HCl (30 mL) was stirred at 100° C.for 16 hours. The mixture was concentrated in vacuo to yield Compound 1(4 g) as a white solid HCl salt. LC-MS: 321 [M+H]⁺.

To a solution of NaOH (1.8 g, 45.2 mmol) in water (100 mL), was addedCompound 1 (4 g, 11.3 mmol) in MeCN (100 mL) dropwise. The mixture wasstirred for 10 minutes at 0° C. Di-t-butyldicarbonate (7.17 g, 33.8mmol) was added and the mixture was stirred for 15 hours at roomtemperature. The resulting mixture was concentrated in vacuo to removeMeCN, then diluted with DCM (300 mL), and the pH adjusted to pH=5-6 with1N aqueous HCl. Then the organic layer was collected and the residue wasextracted with DCM (3×300 mL). The combined organic layers wereconcentrated and washed with hexanes (150 mL) to yield the titlecompound (4 g) as a white solid. LC-MS: 442 [M+Na]⁺.

Preparation 15(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic Acid2,2,3,3,3-pentafluoropropyl Ester

To a solution of(2R,4R)-4-t-Butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (0.9 g, 6 mmol) and 2,2,3,3,3-pentafluoropropan-1-ol (450 mg, 3mmol) in DCM (30 mL) was added DCC (880 mg, 4.3 mmol) and DMAP (260 mg,2.1 mmol). The resulting mixture was stirred for 15 hours at roomtemperature, then concentrated in vacuo. The residue was dissolved inEtOAc (100 mL) and washed with water (30 mL) and saturated aqueous NaCl(30 mL). The organic layer was collected and concentrated and purifiedby column chromatography (hexanes/EtOAc=5:1) to yield Compound 3 (0.4 g)as a white solid. LC-MS: 574 [M+Na]⁺.

A solution of Compound 3 (0.4 g, 690 μmol) in 1.4 M HCl in a 1,4-dixoanesolution (15 mL) was stirred overnight, and then concentrated in vacuo.The residue was dispersed in EtOAc (10 mL), and the precipitate wascollected by filtration to yield the title compound as an off-whitesolid HCl salt (165 mg). LC-MS: 452 [M+H]⁺. ¹H NMR: (DMSO-d₆) 1.95-1.82(m, 2H), 2.99-2.98 (m, 2H), 3.56 (br, 1H), 4.41-4.38 (m, 1H), 4.92-4.82(m, 2H), 6.35 (s, 1H), 7.71-7.38 (m, 8H), 8.09 (s, 3H).

Preparation 16(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic Acid5-Methyl-2-oxo[1,3]dioxol-4-ylmethyl Ester

A suspension of(2R,4R)-4-t-butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (740 mg, 1.8 mmol), 4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (340mg, 1.8 mmol), potassium iodide (58 mg, 350 mmol), and K₂CO₃ (486 mg,3.5 mmol) in DMF (20 mL) was stirred for 4 hours at room temperature.The mixture was diluted with EtOAc (150 mL) and washed with water (30mL). The organic layer was collected and concentrated and purified bycolumn chromatography (hexanes/EtOAc=1:1) to yield a white solid (490mg). LC-MS: 554 [M+23]⁺. A solution of this solid (476 mg, 890 μmol) in3 N HCl in 1,4-dioxane (20 mL) was stirred overnight, and thenconcentrated in vacuo. The residue was dispersed in EtOAc (10 mL), andthe precipitate was collected by filtration to yield the title compoundas an off-white solid (290 mg). LC-MS: 432 [M+H]⁺. ¹H NMR: (DMSO-d₆)1.92-1.82 (m, 2H), 2.16 (s, 3H), 2.99 (br, 2H), 3.56 (br, 1H), 4.35-4.32(m, 1H), 5.017 (s, 2H), 6.17 (s, 1H), 7.39-7.36 (m, 4H), 7.71-7.68 (m,4H), 8.05 (s, 3H).

Preparation 17(2R,4R)-4-Amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic AcidButyryloxymethyl Ester

A solution of(2R,4R)-4-t-butoxycarbonylamino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (900 mg, 2.1 mmol), chloromethyl butyrate (350 mg, 2.6 mmol),sodium iodide (481 mg, 3.21 mmol) and DIPEA (828 mg, 6.42 mmol) in DMF(20 mL) was stirred for 16 hours at 30° C. The mixture was diluted withEtOAc (150 mL) and washed with water (50 mL) and saturated aqueous NaCl(50 mL). The organic layer was collected and concentrated and purifiedby column chromatography (hexanes/EtOAc=5:1) to yield a white solid (240mg). LC-MS: 542 [M+Na]⁺. A solution of this solid (240 mg, 460 μmol) in1.4 M HCl in 1,4-dixoane (15 mL) was stirred overnight, and thenconcentrated in vacuo. The residue was dispersed in EtOAc (10 mL), andthe precipitated was collected by filtration to yield the title compoundas an off-white solid HCl salt (140 mg). LC-MS: 420 [M+H]⁺. ¹H NMR:(DMSO) 0.85 (t, J=7.5 Hz, 3H), 1.61-1.52 (m, 2H), 1.89-1.86 (m, 2H),2.30 (t, J=7.5 Hz, 2H), 2.98 (br, 2H), 3.56 (br, 1H), 4.33-4.30 (m, 1H),5.74-5.68 (m, 2H), 6.21 (s, 1H), 7.37-7.35 (m, 4H), 7.70-7.767 (m, 4H),8.01 (brs, 3H).

Preparation 18(2R,4R)-4-Amino-5-(2′,5′-dichlorobiphenyl-4-yl)-2-hydroxypentanoic AcidEthyl Ester

To a solution of (S)-2-(4-bromobenzyl)-5-oxopyrrolidine-1-carboxylicacid t-butyl ester (33.5 g, 95 mmol) in 1,4-dioxane (1.2 L) was added2,5-dichlorophenylboronic acid (21.7 g, 114 mmol) and Pd(dppf)₂Cl₂ (3.5g, 4.7 mmol) at room temperature under nitrogen. After stirring for 10minutes, a solution of K₂CO₃ (26.1 g, 189 mmol) in water (120 mL) wasadded. The mixture was heated to 60° C. and stirred overnight. Afterevaporation of the solvent, water (400 mL) was added and extracted withEtOAc (3×400 mL). The combined organic layers were washed with saturatedaqueous NaCl (500 mL), dried over anhydrous Na₂SO₄, and concentrated toyield the crude product which was further purified by columnchromatography (hexanes:EtOAc=6:1) to yield Compound 1 (35.8 g) as alight yellow solid. LC-MS: 442 [M+Na].

To a solution of Compound 1 (35.8 g, 85 mmol) in anhydrous DCM (300 mL)was added TFA (30 mL, 405 mmol) at −5° C. under nitrogen. The mixturewas warmed to room temperature and stirred overnight. After evaporationof the solvent, the residue was diluted with EtOAc (500 mL), then washedwith saturated aqueous NaHCO₃ (3×300 mL), water (200 mL), and saturatedaqueous NaCl (250 mL), then dried over Na₂SO₄ and concentrated to yieldcrude Compound 2 (26 g) as a light yellow solid. LC-MS: 320 [M+H].

To a solution of Compound 2 (26 g, 81 mmol) in anhydrous THF (500 mL)was added dropwise n-BuLi in hexane (39 mL, 97 mmol) over 1 hour at −78°C. under nitrogen. After stirring at −78° C. for 2 hours, the reactionwas quenched by adding pivaloyl chloride (12.7 g, 105 mmol) dropwiseover 30 minutes. After stirring at −78° C. for 2 hours, the reaction wasquenched with saturated aqueous NH₄Cl (200 mL) and extracted with EtOAc(3×200 mL). The combined organic layers were washed with saturatedaqueous NaCl (300 mL), dried over anhydrous MgSO₄, filtered andconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=25:1) to yield Compound 3 (33 g) as alight yellow solid. LC-MS: 426 [M+Na].

To a solution of Compound 3 (10 g, 0.025 mol) in anhydrous THF (120 mL)was added dropwise NaHMDS (18.6 mL, 37 mmol) over 30 minutes at −78° C.under nitrogen. After stirring at −78° C. for 2 hours, a solution of(+)-(8,8-dichlorocamphorylsulfonyl)-oxaziridine (11.1 g, 37 mmol) in THF(80 mL) was added dropwise over 30 minutes. After stirring at −78° C.for 2 hours, the reaction was quenched with saturated aqueous NH₄Cl (500mL) and extracted with EtOAc (3×300 mL). The combined organic layerswere washed with saturated aqueous NaCl (300 mL), dried over MgSO₄,filtered and concentrated to yield the crude product which was furtherpurified by chromatography (hexanes:EtOAc=15:1) to yield Compound 4 (4.2g) as a light yellow oil. LC-MS: 442 [M+Na].

A solution of Compound 4 (4.2 g, 10 mmol) in concentrated HCl (80 mL,0.96 mol) was heated at 100° C. for 16 hours. The mixture was thenconcentrated to yield crude the product which was further purified bywashing with Et₂O to yield Compound 5 (3.8 g) as a white solid. LC-MS:354 [M+H].

To a solution of Compound 5 (3.8 g, 10 mmol) in EtOH (5 mL) was added 4MHCl in EtOH (100 mL, 0.4 mol) at room temperature. The mixture washeated at 50° C. for 16 hours. After concentration, the crude productwhich was further purified by washing with Et₂O to yield the titlecompound (3.3 g) as a white solid. LC-MS: 382 [M+H].

Preparation 19(3R,5R)-5-Amino-6-(4-bromo-2-chlorophenyl)-2-ethoxyhex-1-en-3-ol

To a suspension of 4-bromo-2-chlorobenzaldehyde (50 g, 22.8 mmol) inMeOH (500 mL) was added NaBH₄ (17.3 g, 45.6 mmol) in portions at 0° C.The mixture was stirred for 30 minutes and then aqueous NH₄Cl was addedto quench the reaction. The mixture was concentrated in vacuo. Theresidue was extracted with EtOAc (2×200 mL) and the combined organiclayers were dried over anhydrous Na₂SO₄, and concentrated under vacuumto yield Compound 1 (48 g) as a white solid.

To a solution of Compound 1 (46.8 g, 21.1 mmol) in dry DCM (500 mL) wasadded phosphorous tribromide (68.6 g, 25.3 mmol) dropwise at 0° C. undernitrogen. The mixture was stirred for 2 hours and then washed withsaturated aqueous NaHCO₃ (2×200 mL) and saturated aqueous NaCl (200 mL),dried over anhydrous Na₂SO₄, concentrated under vacuum to yield Compound2 (36 g) as a colorless oil.

To a stirred solution of (R)-pyrrolidine-2-carboxylic acid (57.7 g, 0.5mol) and KOH (84 g, 1.5 mol) in isopropyl alcohol (330 mL) was addedbenzyl chloride (70 mL, 0.6 mol) dropwise at 0° C. over 3 hours. Themixture was then stirred overnight at the same temperature. Theresulting mixture was neutralized with concentrate HCl to pH=6, followedby the addition of chloroform (200 mL). The mixture was stirred for 30minutes, then filtered and the precipitate was washed with chloroform(3×100 mL). The combined chloroform solutions were dried over anhydrousNa₂SO₄, and concentrated under vacuum to yield Compound 3 (52 g) as awhite solid. LC-MS: 206 [M+H]⁺.

To a solution of Compound 3 (10 g, 48.8 mmol) in dry DCM (50 mL) wasadded SO₂Cl₂ (7.3 g, 61 mmol) at −20° C. under nitrogen. The mixture wasstirred at −20° C. for 3 hours followed by the addition of a solution of(2-aminophenyl)(phenyl)methanone (6 g, 30.5 mmol) in dry DCM (25 mL) andthe mixture was stirred overnight at room temperature. A solution ofNa₂CO₃ (10.3 g) in water (40 mL) was added at 0° C. The organic layerwas separated and the aqueous layer was extracted with DCM (50 mL×3).The combined organic layers were dried over anhydrous Na₂SO₄ andconcentrated under vacuum. The residue was washed with MTBE (2×50 mL) toyield Compound 4 (8.5 g) as a yellow solid. LC-MS: 385 [M+H]⁺.

To a solution of Compound 4 (29.4 g, 76.5 mmol), glycine (28.7 g, 382.4mmol) and Ni(NO₃)₂.6H₂O (44.5 g, 152.9 mmol) in MeOH (280 mL) was addeda solution of KOH (30 g, 535.3 mmol) in MeOH (100 mL) at 45° C. undernitrogen. The mixture was stirred at 60° C. for an hour. The resultingsolution was neutralized with AcOH (31 mL) and poured into ice water(380 mL). The resulting solid was filtered and dissolved in DCM (450mL), which was washed with saturated aqueous NaCl (150 mL), dried overanhydrous Na₂SO₄ and concentrated. The residue was washed with EtOAc(2×50 mL) to yield compound 5 (38 g) as a red solid. LC-MS: 498 [M+H]⁺.

Compound 5 (14.3 g, 28.7 mmol) and NaOH (3.4 g, 81.6 mmol) were added toa flask which was purged with nitrogen twice. Anhydrous DMF (100 mL) wasadded and the mixture was stirred for 5 minutes at 0° C. before asolution of compound 2 (8.6 g, 30.1 mmol) in DMF (20 mL) was added. Thereaction mixture was stirred at room temperature for 30 minutes untilcomplete consumption of compound 4 (checked by TLC). The resultingmixture was poured into a 5% AcOH aqueous solution (120 mL) which wasthen extracted with DCM (3×150 mL) and the combined organic layers werewashed with saturated aqueous NaCl (150 mL), dried over anhydrous Na₂SO₄and concentrated under vacuum. The residue was recrystallized withDCM/Et₂O (1:1) to yield Compound 6 (15.5 g) as a red solid. LC-MS: 702[M+H]⁺.

To a solution of Compound 6 (46 g, 65.6 mmol) in MeOH (300 mL) was added3N HCl (200 mL). The mixture was refluxed until the red color turnedgreen. The resulting solution was concentrated under vacuum andconcentrated NH₃H₂O (100 mL) was added, and followed by the extractionwith DCM (2×200 mL). The aqueous phase was concentrated under vacuum andsubjected to the cation exchange resin (eluted with NH₃H₂O/EtOH, 1:1) toyield Compound 7 (15 g) as a white solid. LC-MS: 280 [M+H]⁺.

To a suspension of Compound 7 (15 g, 53.9 mmol) in MeCN (150 mL) wasadded a solution of NaOH (4.3 g, 107.7 mmol) in water (150 mL) at 0° C.,and followed by the addition of (BOC)₂O (17.6 g, 80.8 mmol). The mixturewas stirred overnight at room temperature. The resulting solution wasconcentrated under vacuum, followed by the extraction with DCM (2×150mL). The aqueous phase was acidified with 1N HCl to pH=3 and extractedwith EtOAc (3×150 mL). The combined organic layers were washed withsaturated aqueous NaCl (150 mL), dried over anhydrous Na₂SO₄ andconcentrated under vacuum to yield Compound 8 (12.3 g, 60%) as a whitesolid. LC-MS: 402 [M+Na]⁺.

To a suspension of Compound 8 (18.4 g, 48.5 mmol) and Meldrum's acid(8.4 g, 58.2 mmol) in DCM (400 mL) was added DMAP (9.5 g, 77.6 mmol) at−5° C. After stirring for 10 minutes, a solution of DCC (12 g, 58.2mmol) in DCM (100 mL) was added dropwise at −5° C. The mixture wasstirred overnight at room temperature. The resulting solution was cooledto 0° C. and filtered. The filtrate was washed with aqueous citric acid(3×200 mL) and saturated aqueous NaCl (200 mL), dried over anhydrousNa₂SO₄, and concentrated under vacuum. The residue was washed with Et₂O(2×50 mL) to yield Compound 9 (22 g) as a light yellow solid.

To a solution of Compound 9 (22 g, 43.6 mmol) in DCM (400 mL) was addedAcOH (28.8 g, 479.4 mmol) at 0° C. After stirring for 10 minutes, NaBH₄(4.1 g, 109 mmol) was added in portions. The mixture was stirred for anhour at 0° C. The resulting solution was washed with saturated aqueousNaHCO₃ (2×200 mL) and saturated aqueous NaCl (200 mL), dried overanhydrous Na₂SO₄ and concentrated under vacuum. The residue was washedwith ether (2×100 mL) to yield Compound 10 (18.6 g) as an off-whitesolid. LC-MS: 514 [M+Na]⁺.

A solution of Compound 10 (18.6 g, 37.9 mmol) in toluene (350 mL) washeated under reflux for 2 hours. Upon cooling, the mixture wasevaporated to dryness to yield Compound 11 (14 g) as a yellow syrup.LC-MS: 334 [MtBu+H]⁺.

To a solution of Compound 11 (14 g, 36.0 mmol) in DCM (250 mL) was addedTFA (20 mL). The mixture was stirred for 4 hours at 0° C. The resultingsolution was concentrated under vacuum to remove TFA. The residue wasdissolved in DCM (400 mL) and washed with saturated aqueous NaHCO₃(2×200 mL), dried over anhydrous Na₂SO₄ and concentrated to yieldCompound 12 (10 g) as a yellow solid. LC-MS: 290 [M+H]⁺.

To a solution of Compound 12 (10 g, 34.7 mmol) in dry THF (250 mL) wasadded NaH (2.4 g, 69.3 mmol, 70%) at 0° C. The mixture was stirred forone hour at 0° C. under nitrogen. Then pivaloyl chloride (5 g, 41.6mmol) was added. After stirring for another 2 hours, saturated aqueousNaHCO₃ (100 mL) was added to quench the reaction. The resulting mixturewas concentrated and extracted with EtOAc (3×100 mL) and the combinedorganic layers were washed with saturated aqueous NaCl (100 mL), driedover anhydrous Na₂SO₄ and concentrated under vacuum. The residue waspurified by silica gel chromatography (hexanes/EtOAc, 5:1) to yieldCompound 13 (11.8 g) as a white solid. LC-MS: 374 [M+H]⁺.

To a solution of Compound 13 (11.8 g, 31.8 mmol) in dry THF (70 mL) wasadded NaHMDS (24 mL, 47.7 mmol, 2.0 M in THF) dropwise at −78° C. undernitrogen. After stirring for 30 minutes, a solution of(+)-(8,8-dichlorocamphorylsulfonyl)oxaziridine (15.2 g, 50.8 mmol) inTHF (70 mL) was added dropwise at −78° C. The mixture was stirred foranother hour at the same temperature before aqueous NH₄Cl (70 mL) wasadded to quench the reaction. The resulting mixture was extracted withEtOAc (3×150 mL) and the combined organic layers were washed withsaturated aqueous NaCl (150 mL), dried over anhydrous Na₂SO₄,concentrated under vacuum and purified by silica gel chromatography(hexanes/EtOAc, 20:1˜5:1) to yield the crude product (5 g), which wasfurther purified by preparative HPLC to yield Compound 14 (4 g) as ayellow solid. LC-MS: 390 [M+H]⁺.

A solution of Compound 14 (4 g, 10.3 mmol) in concentrated HCl (50 mL)was heated under reflux overnight. The mixture was concentrated undervacuum and the resulting solid was washed with Et₂O (2×50 mL) to yieldCompound 15 (3.1 g) as a white solid HCl salt. LC-MS: 324 [M+H]⁺.

A solution of Compound 15 (3.1 g, 8.6 mmol) in HCl/EtOH (6.7M, 40 mL)was stirred overnight at 50° C. The resulting mixture was concentratedunder vacuum and the residue was washed with ether (2×50 mL) to yieldthe title compound (2.9 g) as an off-white solid HCl salt. LC-MS: 352[M+H]⁺. ¹H NMR: (CD₃OD) 1.268 (t, J=6.9 Hz, 3H), 1.862-1.946 (m, 1H),2.068-2.143 (m, 1H), 3.104-3.199 (m, 2H), 3.769-3.809 (m, 1H),4.162-4.209 (m, 2H), 4.274-4.881 (m, 1H), 7.325 (dd, J=8.1, 2.1 Hz, 1H),7.522 (dd, J=8.3, 3.0 Hz, 1H), 7.696 (d, J=1.8 Hz, 1H).

Preparation 20[(R)-1-Biphenyl-4-ylmethyl-2-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-2-oxoethyl]carbamicAcid t-Butyl Ester

To a solution of (R)-3-biphenyl-4-yl-2-t-butoxycarbonylamino-propionicacid (50 g, 146 mmol), Meldrum's acid (23.3 g, 161 mmol) and DMAP (27.8g, 227 mmol) in anhydrous DCM (500 mL) was added a solution of DCC (33.3g, 161 mmol) in anhydrous DCM (200 mL) over 1 hour at −5° C. undernitrogen. The mixture was stirred at −5° C. for 8 hours, thenrefrigerated overnight, during which tiny crystals of dicyclohexylureaprecipitated. After filtration, the mixture was washed with 5% KHSO₄(4×200 mL) and saturated aqueous NaCl (1×200 mL), then dried underrefrigeration with MgSO₄ overnight. The solution was evaporated to yieldthe title compound (68 g, light yellow solid), which was used withoutfurther purification. LC-MS: 490 [M+Na], 957 [2M+Na].

Preparation 21(2R,4S)-5-biphenyl-4-yl-4-t-butoxycarbonylamino-2-hydroxymethylpentanoicAcid Ethyl Ester (compound 6) and(2S,4S)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-hydroxymethylpentanoicAcid Ethyl Ester (compound 7)

To a solution of crude[(R)-1-biphenyl-4-ylmethyl-2-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-2-oxoethyl]carbamicacid t-butyl ester (68 g, 147 mmol) in anhydrous DCM (1 L) was addedAcOH (96.7 g, 1.6 mol) at −5° C. under nitrogen. The mixture was stirredat −5° C. for 0.5 hour, then NaBH₄ (13.9 g, 366 mmol) was added in smallportions over 1 hour. After stirring for another 1 hour at −5° C.,saturated aqueous NaCl (300 mL) was added. The organic layer was washedwith saturated aqueous NaCl (2×300 mL) and water (2×300 mL), dried overMgSO₄, filtered, and evaporated to yield the crude product, which wasfurther purified by chromatography (hexanes:EtOAc=5:1) to yield Compound1 (46 g, light yellow solid). LC-MS: 476 [M+Na], 929 [2M+Na].

A solution of Compound 1 (46 g, 101 mmol) in anhydrous toluene (300 mL)was refluxed under nitrogen for 3 hours. After evaporation of thesolvent, the residue was purified by chromatography (hexanes:EtOAc=10:1)to yield Compound 2 (27 g, light yellow solid). LC-MS: 374 [M+Na], 725[2M+Na]. ¹H NMR (300 MHz, CDCl3): δ7.64-7.62 (m, 4H), 7.51-7.46 (m, 2H),7.42-7.39 (m, 1H), 7.39-7.30 (m, 2H), 4.50-4.43 (m, 1H), 3.27-3.89 (m,1H), 2.88-2.80 (m, 1H), 2.48-2.42 (m, 2H), 2.09-1.88 (m, 2H), 1.66 (s,9H).

A mixture of Compound 2 (27 g, 77 mmol) andt-butoxy-N,N,N′,N′-tetramethylmethanediamine (40.3 g, 231 mmol) washeated to 80° C. under nitrogen. After stirring for 3 hours at 80° C.,the mixture was diluted with EtOAc (300 mL), washed with water (2×150mL) and saturated aqueous NaCl (2×150 mL), dried over MgSO₄, filtered,and evaporated to yield crude Compound 3 (29.7 g, light yellow oil).LC-MS: 425 [M+H], 835 [2M+H].

To a solution of crude Compound 3 (29.7 g, 73 mmol) in THF (200 mL) wasadded 1 M HCl (81 mL) at 0° C. under nitrogen. After stirring for 1 hourat room temperature, the mixture was diluted with EtOAc (100 mL) andadjusted with saturated aqueous NaHCO₃ to pH 7. The aqueous layer wasextracted with EtOAc (2×150 mL) and the combined organic layers werewashed with water (2×150 mL) and saturated aqueous NaCl(1×150 mL), driedover MgSO₄, filtered, and evaporated to yield crude Compound 4 (29.4 g,yellow oil). LC-MS: 402 [M+Na], 781 [2M+Na].

To a solution of Compound 4 (29.4 g, 77 mmol) in anhydrous THF (300 mL)was added anhydrous EtOH (30 mL) and AcOH (92.5 g, 1.5 mol) at −5° C.under nitrogen. The mixture was stirred at −5° C. for 0.5 hour, thenNaBH₃CN (19.4 g, 308 mmol) was added in small portions over 1 hour.After stirring for one additional hour at −5° C., the mixture wasadjusted with saturated aqueous NaHCO₃ to pH 7. The aqueous layers wereextracted with EtOAc (2×200 mL) and the combined organic layers werewashed with water (2×150 mL) and saturated aqueous NaCl (1×150 mL),dried over MgSO₄, filtered, and concentrated to yield the crude product,which was further purified by chromatography (hexanes:EtOAc=5:1) toyield Compound 5 (11.2 g, light yellow solid). LC-MS: 404 [M+Na], 785[2M+Na].

To a solution of Compound 5 (11.2 g, 29 mmol) in anhydrous EtOH (500 mL)was added anhydrous K₂CO₃ (8.0 g, 58 mmol) at 0° C. under nitrogen.After stirring for 1 hour at 0° C., the mixture was warmed to roomtemperature and stirred for 16 hours. After filtration, the filtrate wasconcentrated and the residual was diluted with water (150 mL), DCM (200mL) and saturated aqueous NaCl (50 mL). After separation, the aqueouslayer was extracted with DCM (2×150 mL). The combined organic layerswere washed with saturated aqueous NaCl (2×200 mL), dried over MgSO₄,and concentrated to yield the crude product which was further purifiedby column chromatography (hexanes:EtOAc=5:1) to yield Compounds 6 and 7(8.3 g, light yellow solid).

Compound 6: LC-MS: 450 [M+Na], 877 [2M+Na]. ¹H NMR (300 MHz, CDCl3):δ7.58-7.23 (m, 9H), 4.46-4.43 (d, 1H), 4.20-4.13 (m, 2H), 3.94 (s, 1H),3.82-3.70 (m, 2H), 2.85-2.70 (m, 3H), 2.25-2.22 (d, 1H), 2.01-1.92 (m,1H), 1.47 (s, 9H), 1.26-1.24 (m, 3H).

Compound 7: LC-MS: 450 [M+Na], 877 [2M+Na]. ¹H NMR (300 MHz, CDCl3):δ7.58-7.55 (m, 4H), 7.50-7.43 (m, 2H), 7.40-7.30 (m, 1H), 7.26-7.23 (m,1H), 4.46 (m, 1H), 4.21-4.13 (m, 2H), 3.94 (m, 1H), 3.82-3.77 (m, 2H),2.83-2.81 (d, 2H), 2.66-2.63 (m, 1H), 2.24 (m, 1H), 1.83-1.81 (m, 2H),1.38 (s, 9H), 1.30-1.25 (m, 3H).

Preparation 22(2S,4S)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-hydroxymethylpentanoicAcid (R⁷=H; P²=BOC) and(2S,4S)-4-Amino-5-biphenyl-4-yl-2-hydroxymethylpentanoic Acid EthylEster (R⁷=—CH₂CH₃; P² Removed)

(2S,4S)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-hydroxymethyl-pentanoicacid ethyl ester (210 mg) was saponified with LiOH to yield theBOC-protected acid (R⁷=H; P²=BOC) (120 mg).(2S,4S)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-hydroxymethyl-pentanoicacid ethyl ester (˜180 mg) was subjected to HCl deprotection to yieldthe amine ester (R⁷=—CH₂CH₃; P² removed) as an HCl salt (120 mg).

Preparation 23(2S,4S)-4-Amino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethylpentanoic AcidEthyl Ester

To a solution of (S)-2-(4-bromobenzyl)-5-oxopyrrolidine-1-carboxylicacid t-butyl ester (18.4 g, 52 mmol) in 1,4-dioxane (500 mL) was added2-fluorophenylboronic acid (8.7 g, 63 mmol) and Pd(dppf)₂Cl₂ (3.8 g, 5.2mmol) at room temperature under nitrogen. After stirring for 10 minutes,a solution of K₂CO₃ (14.4 g, 104 mmol) in water (50 mL) was added. Themixture was heated to 80° C. and stirred at this temperature for 5hours. After evaporation of the solvent, water (300 mL) was added andthe mixture was extracted with EtOAc (3×200 mL). The combined organiclayers were washed with saturated aqueous NaCl (400 mL), dried overNa₂SO₄ and concentrated to give the crude product which was furtherpurified by column chromatography (hexanes:EtOAc=8:1) to yield Compound1 (17.3 g) as a red oil. LC-MS: 392 [M+Na].

A mixture of Compound 1 (17.3 g, 46.7 m mol) andt-butoxy-N,N,N′,N′-tetramethylmethanediamine (24.4 g, 140 mmol) washeated to 80° C. under nitrogen. After stirring for 3 hours at 80° C.,the mixture was diluted with EtOAc (300 mL) and washed with water (2×150mL), saturated aqueous NaCl (150 mL), dried over MgSO₄, filtered, andevaporated to yield crude Compound 2 (20.6 g) as a red oil. LC-MS: 425[M+H], 849 (2M+H).

To a solution of crude Compound 2 (20.6 g, 48.6 mmol) in THF (300 mL),was added 1 M HCl (58 mL, 58 mmol) at 0° C. under nitrogen. Afterstirring for 1 hour at room temperature, the mixture was diluted withEtOAc (100 mL) and adjusted with saturated aqueous NaHCO₃ to a pH of 7.The aqueous layer was extracted with EtOAc (2×150 mL) and the combinedorganic layers were washed with water (2×150 mL) and saturated aqueousNaCl (150 mL), dried over MgSO₄, filtered, and evaporated to yield thecrude Compound 3 (18.9 g) as a red oil. LC-MS: 420 (M+Na), 817 (2M+Na).

To a solution of crude Compound 3 (18.9 g, 47.6 mmol) in anhydrous THF(400 mL) was added anhydrous EtOH (50 mL) and AcOH (57.2 g, 952 mmol) at−5° C. under nitrogen. The mixture was stirred at −5° C. for 30 minutes,then NaBH₃CN (15 g, 238 mmol) was added in small portions over 1 hour.After stirring for an additional 1 hour at −5° C., the mixture wasadjusted with saturated aqueous NaHCO₃ to a pH of 7. The aqueous layerwas extracted with EtOAc (3×200 mL) and the combined organic layers werewashed with water (2×150 mL) and saturated aqueous NaCl (150 mL), driedover MgSO₄, filtered, and concentrated to yield the crude product whichwas further purified by chromatography (hexanes:EtOAc=6:1) to yieldCompound 4 (7.1 g) as a light yellow solid. LC-MS: 422 (M+Na), 821(2M+Na).

To a solution of Compound 4 (7.1 g, 17.7 mmol) in anhydrous EtOH (500mL) was added anhydrous K₂CO₃ (9.8 g, 70.8 mmol) at 0° C. undernitrogen. After stirring for 1 hour at 0° C., the mixture was warmed toroom temperature and stirred for 16 hours. After filtration, thefiltrate was concentrated and the residual was diluted with water (150mL), DCM (200 mL) and saturated aqueous NaCl (50 mL). After separation,the aqueous layer was extracted with DCM (2×150 mL). The combinedorganic layers were washed with saturated aqueous NaCl (2×200 mL), driedover MgSO₄, and concentrated to yield the crude product which wasfurther purified by column chromatography (hexanes:EtOAc=5:1) to yieldCompound 5 (2 g) as a light yellow solid. 2.1 g of the (R,S) isomer wasalso obtained as a light yellow solid.

Compound 5 (400 mg, 0.9 mmol) was dissolved in MeCN (3 mL) and 4 M HClin dioxane (0.5 mL). The mixture was stirred at room temperature for 1hour then concentrated to yield the title compound as an HCl salt (340mg), which was formed as an oil and solidified overnight.

Preparation 24[(R)-2-Biphenyl-4-yl-1-(2,2,5-trimethyl-4,6-dioxo-1,3-dioxinan-5-ylmethyl)ethyl]carbamicAcid t-Butyl Ester

AcOH (8.6 mL) was added to a solution of crude[(R)-1-biphenyl-4-ylmethyl-2-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-2-oxo-ethyl]-carbamicacid t-butyl ester (6.4 g, 14 mmol) in anhydrous MeCN (90 mL) was addedAcOH (8.6 mL) at −5° C. under nitrogen. The mixture was stirred at −5°C. for 30 minutes, then sodium borohydride (1.3 g, 34.5 mmol) was addedin small portions over 2 hours. After stirring for another 1 hour at −5°C., saturated aqueous NaCl and 1.7 M of NaCl in water (30 mL) was added.The layers were separated and the organic layer was washed withsaturated aqueous NaCl (2×30 mL) and water (2×30 mL), dried under MgSO₄,filtered and evaporated. The resulting crude product was furtherpurified by chromatography (5:1 heptane:EtOAc) to yield[(S)-2-biphenyl-4-yl-1-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-ylmethyl)ethyl]carbamicacid t-butyl ester (1.1 g, purity 98.4%) as a light yellow solid.

[(S)-2-Biphenyl-4-yl-1-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-ylmethyl)-ethyl]carbamicacid t-butyl ester (5.0 g, 11 mmol) and K₂CO₃ (1.8 g, 13.2 mmol) weredissolved in DMF (33.9 mL) and cooled to 0° C. with stirring undernitrogen. Methyl iodide (892 μL) was added and the resulting mixture wasstirred at 0° C. for 1 hour. The mixture was allowed to warm to roomtemperature. Saturated aqueous NaCl (35 mL) and EtOAc (35 mL) wereadded, and the resulting mixture was stirred for 2 minutes. The layerswere separated and the organic layer was evaporated. The residue wastriturated with EtOAc (20 mL). The solid was filtered off and driedunder vacuum. The filtrate was concentrated and triturated again withEtOAc to yield the title compound (3.9 g).

Preparation 25(2S,4R)-5-Biphenyl-4-yl-4-t-butoxycarbonylamino-2-hydroxymethyl-2-methylpentanoicAcid (P²=BOC) and(2S,4R)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-2-methylpentanoic Acid(P² Removed)

Distilled Water (140 mL) was purged 30 minutes under nitrogen, thencannulated into a vessel containing 0.1 M of samarium diiodide in THF(800 mL), exercising caution not to allow any air to come into contactwith solution. While maintaining an atmosphere of nitrogen, a degassedsolution of[(R)-2-biphenyl-4-yl-1-(2,2,5-trimethyl-4,6-dioxo-1,3-dioxinan-5-ylmethyl)ethyl]carbamicacid t-butyl ester (3.7 g, 8.0 mmol) and THF (100 mL) was added viacanula. The resulting mixture was stirred for 15 minutes, then exposedto air. Saturated aqueous NaCl (12 mL), 10% citric acid (6 mL), andEtOAc (30 mL) were added. The mixture was stirred for 5 minutes, thenboth layers were extracted. The organic layer was dried over Na₂SO₄ andconcentrated under vacuum. The crude product was purified bychromatography (330 g gold column, 50% EtOAc with 0.5% AcOH/ethergradient) to yield the BOC-protected acid. (P²=BOC) (1.4 g). TheBOC-protected acid was dissolved in MeCN (10 mL), followed by theaddition of 4N HCl in dioxane (10 mL). The solvent was evaporated andthe product azeotroped with toluene (2×) to yield the acid. (P² removed)(1.0 g).

Preparation 26(2S,4R)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-2-methylpentanoic AcidEthyl Ester

(2S,4R)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-2-methylpentanoic acid(0.3 g, 957 μmol) was combined with EtOH (6 mL) and 4 M of HCl in1,4-dioxane (718 μL), and stirred overnight. The solvents wereevaporated and the product was azeotroped with toluene (2×) to yield thetitle compound (295 mg), which was used without further purification.

Preparation 27[(R)-1-(3′-Fluorobiphenyl-4-ylmethyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-ethyl]carbamicAcid t-Butyl Ester

To a solution of (R)-2-amino-3-(4-bromophenyl)propionic acid (50 g, 0.2mol) in MeCN (700 mL) was added a solution of NaOH (16.4 g, 0.4 mol) inwater (700 mL) at −5° C. After stirring for 10 minutes, a solution of(BOC)₂O (44.7 g, 0.2 mol) in MeCN (100 mL) was added. The mixture waswarmed to room temperature and stirred overnight. After evaporation ofthe MeCN, the residue was diluted with DCM (800 mL) and acidified with 1M HCl to pH 2 at −5° C. The aqueous layer was extracted with DCM (3×200mL). The combined organic layers were washed with saturated aqueous NaCl(500 mL), dried over anhydrous Na₂SO₄ and concentrated to yield Compound1 (64.2 g, white solid). LC-MS: 366 [M+Na], 709 [2M+Na].

To a solution of Compound 1 (64.2 g, 187 mmol) in 1,4-dioxane (500 mL)was added 3-fluorophenylboronic acid (31.3 g, 224 mmol) and Pd(dppf)₂Cl₂(13.7 g, 19 mmol) at room temperature under nitrogen. After stirring for10 min, a solution of K₂CO₃ (51.7 g, 374 mmol) in water (250 mL) wasadded. The mixture was heated to 100° C. and stirred overnight. Afterevaporation of the solvent, water (200 mL) was added. The aqueous layerwas acidified with 1 M HCl to pH 2 and extracted with EtOAc (3×200 mL).The combined organic layers were washed with saturated aqueous NaCl (400mL), dried over anhydrous Na₂SO₄, and concentrated to yield the crudeproduct which was further purified by column chromatography(hexanes:EtOAc=4:1) to yield Compound 2 (45 g, light yellow oil). LC-MS:382 [M+Na], 741 [2M+Na].

To a solution of Compound 2 (45 g, 125 mmol), Meldrum's acid (23.5 g,163 mmol), and DMAP (26.0 g, 213 mmol) in anhydrous DCM (500 mL) wasadded a solution of DCC (33.3 g, 163 mmol) in anhydrous DCM (200 mL)over 1 hour at −5° C. under nitrogen. The mixture was stirred at −5° C.for 8 hours, then refrigerated overnight, during which tiny crystals ofdicyclohexylurea precipitated. After filtration, the mixture was washedwith 5% KHSO₄ (4×200 mL) and saturated aqueous NaCl (1×200 mL), thendried under refrigeration with anhydrous MgSO₄ overnight. The solutionwas evaporated to yield the crude Compound 3 (57.7 g, light yellow oil).LC-MS: 508 [M+Na], 993 [2M+Na].

To a solution of the crude Compound 3 (57.7 g, 119 mmol) in anhydrousDCM (1 L) was added AcOH (78.4 g, 1.3 mol) at −5° C. under nitrogen. Themixture was stirred at −5° C. for 0.5 hour, then NaBH₄ (11.3 g, 0.3 mol)was added in small portions over 1 hour. After stirring for a another 1hour at −5° C., saturated aqueous NaCl (300 mL) was added. The organiclayer was washed with saturated aqueous NaCl (2×300 mL) and water (2×300mL), dried over anhydrous MgSO₄, filtered and concentrated to yield thecrude product, which was further purified by chromatography(hexanes:EtOAc=6:1) to yield Compound 4 (28 g, light yellow oil). LC-MS:494 [M+Na], 965 [2M+Na].

To a solution of Compound 4 (28 g, 60 mmol) in anhydrous DMF (250 mL)was added K₂CO₃ (9.9 g, 72 mmol) and CH₃I (25.6 g, 180 mmol) at 0° C.under nitrogen. After stirring for 1 hour at 0° C., the mixture waswarmed to room temperature and stirred overnight. The mixture wasdiluted with water (3 L) and extracted with EtOAc (3×300 mL). Thecombined organic layers were washed with saturated aqueous NaCl (500mL), dried over anhydrous Na₂SO₄, and concentrated to give the crudeproduct which was further purified by chromatography (hexanes:EtOAc=5:1)to yield the title compound (11.7 g, light yellow solid). LC-MS: 508[M+Na], 993 [2M+Na]. ¹H NMR (300 MHz, CD₃OD): δ7.52-7.49 (m, 2H),7.41-7.39 (m, 2H), 7.32-7.27 (m, 3H), 7.07-7.01 (m, 1H), 6.21-6.18 (d,1H), 3.79 (m, 1H), 2.78-2.61 (m, 2H), 2.35-2.20 (m, 2H), 1.76 (s, 6H),1.59 (s, 3H), 2.21 (s, 1H), 1.28 (s, 9H).

Preparation 28(2S,4R)-4-t-Butoxycarbonylamino-5-(3′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid (P²=BOC) and(2S,4R)-4-Amino-5-(3′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicAcid (P² Removed)

Distilled Water (181 mL) was purged 1 hour under nitrogen, thencannulated into a vessel containing 0.1 M of samarium diiodide in THF(800 mL). While maintaining an atmosphere of nitrogen, a similarlydegassed solution of[(R)-1-(3′-fluorobiphenyl-4-ylmethyl)-2-(2,2,5-trimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-ethyl]-carbamicacid t-butyl ester (4.9 g, 10.0 mmol) and THF (20 mL) was added viacanula. The resulting mixture was stirred for 15 minutes, then exposedto air. The solvent was evaporated, and EtOAc (200 mL), saturatedaqueous NaCl (50 mL) and 10% citric acid (20 mL) were added. The mixturewas stirred for 5 minutes, then both layers were extracted. The organiclayer was dried over Na₂SO₄ and concentrated under vacuum. The crudeproduct was purified by chromatography (330 g gold column, 1:1ether:EtOAc with 0.5% AcOH) to yield the BOC-protected acid. (P²=BOC)(1.5 g). A portion of the BOC-protected acid was dissolved in 4M HCl indioxane (6 mL) and MeCN (10 mL). The solvent was evaporated under vacuumto yield the acid (P² removed).

Preparation 293-(N-Biphenyl-4-ylmethyl-N′-t-butoxycarbonylhydrazino)-2-hydroxy-2-methylpropionicAcid Methyl Ester

4-(Bromomethyl)biphenyl (2.00 g, 8.09 mmol) and DIPEA (1.4 mL, 8.1 mmol)were dissolved in DMF (40.0 mL), then t-butyl carbazate (2.1 g, 16.2mmol) was added and the mixture was stirred at room temperatureovernight. Upon completion of the reaction, the mixture was partiallyconcentrated, and the residue was partitioned between EtOAc andsaturated aqueous NaHCO₃. The EtOAc layer was dried over Na₂SO₄ andconcentrated. The crude product was purified by flash chromatography(0-60% EtOAc/hexanes with 0.5% DIPEA) to yield Compound 1 (1.3 g.)

Compound 1 (460 mg, 1.5 mmol) was dissolved in isopropyl alcohol (10.0mL), then methyl 2-methylglycidate (180 μL, 1.7 mmol) was added and themixture was heated to 85° C. overnight. Upon completion of the reaction,the mixture was partitioned between EtOAc and saturated aqueous NaHCO₃.The EtOAc layer was then dried over Na₂SO₄ and concentrated to yield thetitle compound (0.5 g), which was used without further purification.

Preparation 30(R)-3-[N-(4-Bromobenzyl)-N′-t-butoxycarbonylhydrazino]-2-hydroxypropionicAcid Methyl Ester

4-Bromobenzyl bromide (5.0 g, 20 mmol) and DIPEA (3.48 mL, 20.0 mmol)were dissolved in DMF (20 mL). t-Butyl carbazate (7.9 g, 60.0 mmol) wasadded and the mixture was stirred at room temperature until the reactionwas complete. The mixture was partially concentrated, then the residuewas partitioned between EtOAc and saturated aqueous NaHCO₃. The EtOAclayer was then dried over Na₂SO₄ and concentrated. The crude product waspurified by flash chromatography to yield Compound 1 (3.8 g).

Compound 1 (1.9 g, 6.3 mmol) was dissolved in isopropyl alcohol (26.4mL). Methyl (2R)-glycidate (1.1 mL, 12.6 mmol) was added and the mixturewas heated at 90° C. until the reaction was complete (˜4 days). Themixture was cooled to room temperature and concentrated to yield thetitle compound (2.5 g) as a white solid.

Preparation 31 (R)-3-[N-(3′-Chlorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionic Acid EthylEster

(R)-3-[N-(4-Bromobenzyl)-N′-t-butoxycarbonylhydrazino]-2-hydroxypropionicacid methyl ester (600 mg, 1 mmol), 3-chlorophenylboronic acid (419 mg,2.7 mmol), and K₂CO₃ (617 mg, 4.5 mmol) were combined in EtOH (5 mL) andwater (1 mL), followed by the addition of SilicaCat® Pd(0) (0.09 mmol/gloading, 1160 mg, 104 μmol). The mixture was heated in a microwavereactor at 120° C. until the reaction was complete (˜30 minutes). Themixture was filtered and concentrated. The residue was dissolved intoMeCN/AcOH and purified by reverse phase chromatography (55 g column;gradient 30-95% MeCN in water with 0.1% TFA). The clean fractions werecollected, concentrated and then dissolved in 4M HCl in dioxane (6 mL)and EtOH (6 mL). The mixture was stirred at room temperature overnight,then concentrated to yield the title compound (250 mg), which was usedwithout further purification.

Preparation 32(S)-2-(1-Biphenyl-4-yl-1-methylethyl)-5-oxo-pyrrolidine-1-carboxylicAcid t-Butyl Ester

To a solution of 2-(4-bromophenyl)acetonitrile (130.0 g, 0.7 mol) andiodomethane (103.9 mL, 1.7 mol) in THF (1.0 L) was added NaH (60%dispersion in mineral oil, 66.7 g, 1.7 mol) in small portions at 10° C.After completion of the addition, the mixture was stirred at 10° C. foranother 2 hours. The mixture was poured into ice water (2.0 L) andextracted with EtOAc (1.5 L). The organic layer was washed withsaturated aqueous NaCl, dried over anhydrous MgSO₄ and concentrated toyield Compound 1 (175 g, containing mineral oil) as a yellow oil, whichwas used directly without further purification. ¹H NMR (CDCl₃, 300 MHz)δ 7.52 (d, J=9.0 Hz, 2H), 7.38 (d, J=9.0 Hz, 2H), 1.72 (s, 6H).

To a solution of Compound 1 (175 g, containing mineral oil) in DCM (1.0L) was added DIBAL (1.0 M solution in DCM, 700 mL, 0.70 mol) dropwise at−78° C. The reaction mixture was stirred at −78° C. for 1.5 hours andthen quenched carefully with 3.0 N HCl (1.0 L). The resulting mixturewas stirred at room temperature overnight and the organic layer waswashed with saturated aqueous NaCl, dried over anhydrous Na₂SO₄ andconcentrated to yield Compound 2 (180 g) as a yellow oil, which was useddirectly without further purification. ¹H NMR (CDCl₃, 300 MHz) δ 9.48(s, 1H), 7.53 (d, J=11.0 Hz, 2H), 7.17 (d, J=11.0 Hz, 2H), 1.46 (s, 6H).

To an aqueous solution of NaCN (32.7 g in 1.0 L of H₂O, 0.7 mol) wereadded (NH₄)₂CO₃ (380 g, 4.0 mol) and Compound 2 (180 g). The reactionmixture was refluxed overnight and then concentrated under reducedpressure at 75° C. Water (350 mL) was added to the residue and themixture was concentrated again. The residue was suspended in petroleumether (700 mL) and water (250 mL) and the resulting mixture was stirredat room temperature for 15 minutes. The precipitate was collected byfiltration and dried to yield Compound 3 (150 g) as a white solid. ¹HNMR (DMSO-d6, 300 MHz) δ 10.39 (s, 1H), 8.05 (s, 1H), 7.48 (d, J=9.0 Hz,2H), 7.28 (d, J=9.0 Hz, 2H), 4.17 (s, 1H), 1.42 (s, 3H), 1.34 (s, 3H).

A suspension of Compound 3 (150 g, 0.51 mol) in 6.0 N NaOH (400 mL) andethane-1,2-diol (300 mL) was stirred at 120° C. for 38 hours. Themixture was cooled to room temperature and neutralized with an HClsolution. The precipitate was collected by filtration and dried to yieldCompound 4 (250 g, containing NaCl salt) as a white solid. ¹H NMR(DMSO-d6, 300 MHz) δ 7.35 (d, J=9.0 Hz, 2H), 7.17 (d, J=9.0 Hz, 2H),3.22 (s, 1H), 1.16 (s, 3H), 1.15 (s, 3H).

To a suspension of Compound 4 (250 g, containing NaCl salt) in MeOH (1.0L) was added thionyl chloride (72.0 mL, 1.0 mol) dropwise at 5° C. Themixture was refluxed overnight and the solvent was removed under reducedpressure. The residue was partitioned between DCM (1.0 L) and saturatedaqueous NaHCO₃ (1.5 L). The organic layer was washed with saturatedaqueous NaCl, dried over anhydrous Na₂SO₄ and concentrated to yield thecorresponding methyl ester (90.0 g). 2-Phenylacetyl chloride (48.6 g,0.32 mol) was added dropwise to a solution of the ester (90.0 g) andEt₃N (56.5 mL, 0.41 mol) in DCM (1.0 L) at 0° C. and the mixture wasstirred at 0° C. for 30 minutes. The mixture was washed with 1.0 N HCl(500 mL) and saturated aqueous NaCl, respectively. The organic layer wasdried over anhydrous Na₂SO₄ and concentrated to yield Compound 5 (120g). ¹H NMR (CDCl₃, 300 MHz) δ 7.32 (m, 5H), 7.18 (m, 2H), 6.95 (m, 2H),5.68 (br s, 1H), 4.76 (d, J=9.0 Hz, 1H), 3.57 (s, 3H), 3.53 (d, J=5.0Hz, 2H), 1.30 (s, 3H), 1.25 (s, 3H).

To a solution of Compound 5 (120 g, 0.30 mol) in MeOH (500 mL) was added4.0 N NaOH (200 mL). The mixture was stirred at room temperature for 4hours and then the pH was adjusted to pH=1 with 3.0 N HCl. The resultingmixture was extracted with EtOAc (2×300 mL). The combined extracts werewashed with saturated aqueous NaCl, dried over anhydrous Na₂SO₄, andconcentrated under reduced pressure. The residue was recrystallized fromEtOAc/hexanes to yield Compound 6 (82.0 g). ¹H NMR (DMSO-d6, 300 MHz) δ7.41 (d, J=6.0 Hz, 2H), 7.22 (m, 5H), 6.99 (d, J=6.0 Hz, 2H), 4.65 (d,J=9.0 Hz, 1H), 3.52 (d, J=14.0 Hz, 1H), 3.36 (d, J=14.0 Hz, 1H), 1.34(s, 3H), 1.30 (s, 3H).

A suspension of Compound 6 (82.0 g, 0.21 mol) in distilled water (3.0 L)was adjusted to pH=8.5 with 3.0 N LiOH and a clear solution was formedImmobilized Penicillinase (20.0 g) was added and the resulting mixturewas stirred at 37° C. for 60 hours. The mixture was filtered and thefiltrate was adjusted to pH=1 with 3.0 N HCl and extracted with EtOAc.The combined extracts were washed with saturated aqueous NaCl, driedover anhydrous Na₂SO₄ and concentrated to yield Compound 7 (59.0 g, 80%ee, containing 2-phenylacetic acid).

A suspension of Compound 7 (59.0 g, containing 2-phenylacetic acid) in6.0 N HCl (500 mL) was refluxed overnight. The mixture was washed withEtOAc (300 mL) and the aqueous phase was concentrated under reducedpressure to yield the corresponding amino acid as its hydrochloridesalt. The salt was dissolved in water (300 mL) and the solution wasadjusted to pH=11. A solution of (BOC)₂O (33.0 g, 0.2 mol) in acetone(200 mL) was added and the mixture was stirred at room temperature for 2hours. The mixture was washed with hexanes (200 mL) and the aqueousphase was adjusted to pH=2. The resulting mixture was extracted withEtOAc (2×300 mL). The combined extracts were washed with saturatedaqueous NaCl, dried over anhydrous Na₂SO₄ and concentrated to yieldCompound 8 (37.0 g) as a white solid. ¹H NMR (CDCl₃, 300 MHz) δ 9.48 (brs, 1H), 7.46 (d, J=7.0 Hz, 2H), 7.26 (d, J=7.0 Hz, 2H), 5.02 (br s, 1H),4.56 (d, J=9.0 Hz, 1H), 1.39 (s, 9H).

A mixture of Compound 8 (37.0 g, 0.1 mol) in dioxane (200 mL) and 1.0 NK₂CO₃ (200 mL) was degassed for 30 minutes with nitrogen, followed bythe addition of phenylboronic acid (13.4 g, 0.1 mol) and Pd(PPh₃)₄ (1.6g, 1.4 mmol). The mixture was heated at 75° C. for 8 hours and thencooled to room temperature. The mixture was washed with EtOAc/hexanes(150 mL, 1:1) and the aqueous phase was adjusted to pH=2 and extractedwith EtOAc (2×300 mL). The combined extracts were washed with saturatedaqueous NaCl, dried over anhydrous Na₂SO₄ and concentrated to yieldCompound 9 (31.0 g, 84% yield) as a white solid.

A solution of Compound 9 (31.0 g, 84 mmol), Meldrum's acid (13.3 g, 92mmol) and DMAP (15.4 g, 0.13 mol) in DCM (400 mL) was cooled to −5° C.and a solution of DCC (19.0 g, 92 mmol) in DCM (200 mL) was added over 1hour. The mixture was stirred at −5° C. overnight. The precipitate wasfiltered off and the filtrate was washed with 1.0 N HCl(2×700 mL) andsaturated aqueous NaCl, respectively. After the organic layer containingCompound 10 was dried over anhydrous MgSO₄, it was used directly for thenext step without concentration.

A solution of Compound 10 in DCM (600 mL) was cooled to −5° C. and AcOH(45.0 mL) was added. Then NaBH₄ (7.0 g, 0.2 mol) was added in smallportions over 30 minutes and the mixture was stirred at −5° C. for 3hours. Water (50.0 mL) was added dropwise followed by addition ofsaturated aqueous NaCl (450 mL). The organic layer was washed with water(2×300 mL) and saturated aqueous NaHCO₃ (2×300 mL), dried over anhydrousMgSO₄ and concentrated to yield Compound 11 (32.0 g, 75% ee) as anoff-white solid. After recrystallization from EtOH, chirally pureCompound 11 (13.0 g) was obtained. ¹H NMR (CDCl₃, 300 MHz) δ 7.61 (m,10H), 4.46 (br s, 1H), 4.26 (m, 1H), 3.72 (br s, 1H), 2.23 (m, 1H), 1.79(s, 3H), 1.76 (s, 3H), 1.48 (s, 6H), 1.39 (s, 9H).

A solution of Compound 11 (13.0 g, 27.0 mmol) in toluene (100.0 mL) wasrefluxed for 3 hours. After evaporation of the solvent, the residue wasrecrystallized from hexanes/EtOAc (3:1) to yield the title compound (8.0g) as a white solid.

Preparation 33(2R,4S)-4-Amino-5-biphenyl-4-yl-2-hydroxy-5-methylhexanoic Acid EthylEster

A mixture of(S)-2-(1-biphenyl-4-yl-1-methylethyl)-5-oxo-pyrrolidine-1-carboxylicacid t-butyl ester (14.0 g, 36.9 mmol, racemic) in a 3.0 N HCl-EtOAcsolution (150 mL) was stirred at room temperature for 3 hours. Thesolvent was removed under reduced pressure to yield Compound 1 (10.0 g)as a white solid.

To a solution of Compound 1 (10.0 g, 35.8 mmol) in THF (80.0 mL) wasadded BuLi (2.5 M in hexanes, 15.0 mL) dropwise at −78° C. After themixture was stirred for 30 minutes pivaloyl chloride (4.8 mL, 39.4 mmol)was added dropwise. The mixture was stirred at −78° C. for 1 hour andthen quenched with saturated aqueous NH₄Cl. The resulting mixture wasextracted with EtOAc and the combined extracts were washed withsaturated aqueous NaCl, dried over anhydrous MgSO₄ and concentrated. Theresidue was purified by flash column chromatography on silica gel toyield Compound 2 (9.0 g) as a white solid.

To a solution of Compound 2 (9.0 g, 24.7 mmol) in THF (50.0 mL) wasadded sodium bis(trimethylsilyl)amide (2.0 M in THF, 18.5 mL, 37.0 mmol)dropwise at −78° C. The mixture was stirred for 20 minutes and asolution of oxaziridine derivative (10.8 g, 37.0 mmol) in THF (30.0 mL)was added dropwise. The mixture was stirred at −78° C. for 30 minutesand then quenched with saturated aqueous NH₄Cl. The resulting mixturewas extracted with EtOAc (1.0 L) and the extract was washed with 1.0 NHCl and saturated aqueous NaCl, dried over anhydrous MgSO₄ andevaporated to remove most of the solvent. The precipitate was filteredoff and the filtrate was concentrated. The residue was purified by flashcolumn chromatography on silica gel (DCM:hexanes=1:1 to DCM) to yieldCompound 3 (4.3 g, racemic). This racemate was subjected to chiralAD-column chromatography to afford chirally pure Compound 3 (1.4 g). ¹HNMR (DMSO-d6, 300 MHz) δ 7.63 (m, 4H), 7.49 (m, 4H), 4.83 (d, 1H), 3.29(m, 1H), 2.31 (m, 2H), 1.40 (s, 3H), 1.36 (s, 3H), 1.28 (s, 9H). LC-MS(ESI): m/z 380.1 [M+H]+.

A solution of Compound 3 (1.7 g, 160 mmol) in EtOH (15.0 mL) and 12.0 NHCl (15.0 mL) was heated at 90˜95° C. for 20 hours. The solvent wasremoved and the residue was treated with a 3.0 N HCl-EtOH solution (25.0mL) under reflux for another 3 hours. After removal of the solvent, theresidue was purified by preparative HPLC to yield the title compound(0.6 g) as a foamy solid HCl salt. ¹H NMR (DMSO-d6, 300 MHz) δ 7.88 (brs, 3H), 7.68 (m, 4H), 7.49 (m, 4H), 7.35 (m, 1H), 6.11 (br s, 1H), 4.11(br s, 1H), 4.05 (q, 2H), 3.61 (br s, 1H), 1.67 (m, 2H), 1.40 (s, 3H),1.36 (s, 3H), 1.09 (t, 3H). LC-MS (ESI): m/z 342.1 [M+H]+.

Preparation 34(2S,4S)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-5-methylhexanoic AcidEthyl Ester

A mixture of(S)-2-(1-biphenyl-4-yl-1-methylethyl)-5-oxo-pyrrolidine-1-carboxylicacid t-butyl ester (8.0 g, 21.2 mmol) andt-butoxy-N,N,N′,N′-tetramethylmethanediamine (10.0 g, 63.6 mmol) washeated at 80° C. for 3 hours. The mixture was cooled to room temperatureand diluted with EtOAc (200 mL). The resulting solution was washed withwater (2×100 mL) and saturated aqueous NaCl, dried over anhydrous MgSO₄and concentrated to yield Compound 1 (9.2 g, quantitative) as an oil. ¹HNMR (CDCl₃, 300 MHz) δ 7.53 (m, 9H), 6.95 (s, 1H), 4.60 (br s, 1H), 2.90(s, 1H), 2.62 (m, 2H), 1.61 (s, 9H), 1.39 (s, 3H), 1.34 (s, 3H).

To a solution of Compound 1 (9.2 g, 21.2 mmol) in THF (80.0 mL) wasadded 1.0 N HCl (25.0 mL) at 0° C. The mixture was stirred at roomtemperature for 2 hours and then diluted with EtOAc (100 mL). Theresulting mixture was neutralized with saturated aqueous NaHCO₃ andextracted with EtOAc (2×100 mL). The combined extracts were washed withwater (2×100 mL) and saturated aqueous NaCl, dried over anhydrous MgSO₄and concentrated to yield Compound 2 (8.6 g, quantitative) as an oil.LC-MS (ESI): m/z 430.1 [M+Na]+.

To a solution of Compound 2 (8.6 g, 21.2 mmol) in THF (150 mL) and EtOH(15.0 mL) was added AcOH (24.3 mL, 0.4 mol) at −5° C. After the mixturewas stirred at −5° C. for 30 minutes, NaBH₃CN (5.3 g, 84.8 mmol) wasadded in small portions over 1 hour. The mixture was stirred at −5° C.for 1 hour and neutralized with saturated aqueous NaHCO₃. The resultingmixture was extracted with EtOAc (2×100 mL). The combined extracts werewashed with water (2×100 mL) and saturated aqueous NaCl, dried overanhydrous MgSO₄ and concentrated to yield Compound 3 (8.67 g,quantitative) as a foamy solid.

To a solution of Compound 3 (3.5 g, 8.6 mmol) in EtOH (30.0 mL) wasadded K₂CO₃ (2.4 g, 17.1 mmol) at 0° C. The mixture was stirred at 0° C.for 1 hour and then allowed to warm to room temperature and stirredovernight. The mixture was filtered and the filtrate was concentrated.The residue was treated with water (20 mL) and the resulting mixture wasextracted with DCM (3×25 mL). The combined extracts were washed withsaturated aqueous NaCl, dried over anhydrous MgSO₄ and concentrated. Theresidue was purified by flash column chromatography on silica gel(hexanes:EtOAc=6:1) to yield Compound 4 (2.2 g) as a foamy solid. ¹H NMR(CDCl₃, 300 MHz) δ 7.53 (m, 9H), 4.35 (br s, 1H), 4.15 (m, 2H), 3.95 (brs, 1H), 3.65 (m, 2H), 2.61 (br s, 1H), 1.79 (m, 1H), 1.45 (s, 9H), 1.35(s, 3H), 1.29 (s, 3H), 1.25 (t, 3H). LC-MS (ESI): m/z 478.2 [M+Na]+.

A mixture of Compound 4 (2.2 g, 4.8 mmol) in a 2.0 N HCl-EtOH solution(30.0 mL) was stirred at room temperature for 3 hours. Removal of thesolvent under reduced pressure yielded the title compound (1.6 g) as afoamy solid HCl salt. ¹H NMR (DMSO-d6, 300 MHz) δ 8.08 (br s, 3H), 7.55(m, 9H), 4.95 (br s, 1H), 3.95 (m, 2H), 3.48 (m, 2H), 2.75 (br s, 1H),1.79 (m, 2H), 1.47 (s, 3H), 1.40 (s, 3H), 1.09 (t, 3H). LC-MS (ESI): m/z356.1 [M+H]+.

Preparation 35 3-[N-(4-Bromobenzyl)hydrazino]-2-hydroxypropionic AcidEthyl Ester

A solution of(R)-3-[N-(4-bromobenzyl)-N′-t-butoxycarbonylhydrazino]-2-hydroxypropionicacid methyl ester (25 g, 62 mmol) in EtOH/HCl (310 mL, 1.0 M, 0.3 mol)was stirred overnight. The mixture was concentrated and the reside waswashed with EtOAc (120 mL) and filtered to yield the title compound as awhite solid HCl salt (15 g).

Preparation 36 Oxalic acid(R)-2-[N-(4-bromobenzyl)-N′-ethoxyoxalylhydrazino]-1-ethoxycarbonylethylEster Ethyl Ester

Ethyl oxalyl chloride (70 μL, 630 μmol) was added dropwise to a solutionof 3-[N-(4-bromobenzyl)hydrazino]-2-hydroxypropionic acid ethyl ester(200 mg, 630 μmol) and Et₃N (220 μL, 1.6 mmol) in DCM (4.0 mL, 62.2mmol) at 0° C. The resulting mixture was stirred for 15 minutes at 0° C.and for 15 minutes at room temperature. Water (3 mL) was added, thelayers were separated, and the aqueous layer was extracted with DCM (2×2mL). The DCM layers were combined, dried over MgSO₄, and concentrated toyield the title compound (275 mg).

Preparation 37 N′-(4-Bromobenzyl)hydrazinecarboxylic Acid t-Butyl Ester

To a stirred solution of t-butyl carbazate (50 g, 0.4 mol) in dry THF(400 mL) was added dropwise a solution of 4-bromobenzaldehyde (70 g, 0.4mol) in dry THF (200 mL). The mixture was stirred at room temperaturefor 2 hours, and then concentrated in vacuo to yield Compound 1 as ayellow solid (113.8 g). LC-MS: 243 [M-tBu+H]⁺.

To a solution of Compound 1 (113.8 g, 0.4 mol) in dry THF (1 L) wasadded NaCNBH₃ (36 g, 0.6 mol) in portions at 0° C. AcOH (180 mL) wasadded dropwise and the resulting mixture was stirred at room temperatureovernight. Water (2 L) and EtOAc (1.5 L) were added and the aqueousphase was adjusted to pH=7 with a saturated aqueous Na₂CO₃ solution. Theorganic layer was separated, washed with saturated aqueous NaCl andwater (200 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo.The residue was treated with MeOH (2 L) and 1N NaOH (1.5 L), and thenstirred at room temperature for 2 hours. After the removal of the MeOHsolvent, the precipitate was collected by filtration to yield the titlecompound as a white solid (112 g). LC-MS: 245 [M-tBu+H]⁺.

Preparation 38(R)-3-[N′-t-Butoxycarbonyl-N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicAcid Methyl Ester

To a solution of N′-(4-bromobenzyl)hydrazinecarboxylic acid t-butylester (60 g, 0.2 mol) in 1,4-dioxane (1.5 mL) was added5-chloro-2-fluorophenylboronic acid (38 g, 0.2 mol) and Pd(dppf)Cl₂ (7.3g). The mixture was stirred at room temperature under nitrogen for 10minutes, and then, K₂CO₃ (55.2 g, 0.4 mol) in water (240 mL) was added.The resulting mixture was stirred at 60° C. for 3 hours, and then cooledto room temperature and concentrated in vacuo. The residue was extractedwith EtOAc (3×300 mL). The combined organic layers were dried overanhydrous Na₂SO₄ and concentrated in vacuo. The product was purified bycolumn chromatography (PE:EtOAc=10:1˜5:1) to yield Compound 1 as a pinksolid (56 g). LC-MS: 701 [2M+H]⁺

To a solution of Compound 1 (20 g, 57 mmol) in isopropyl alcohol (250mL) was added methyl (2R)-glycidate (8.7 g, 86 mmol) under nitrogen. Themixture was stirred at 85° C. for 3 days, then cooled to roomtemperature. The precipitated solid was collected by filtration to yieldthe title compound as an off-white solid (18.5 g). LC-MS: 397[M-tBu+H]⁺.

Preparation 39(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicAcid Ethyl Ester

A solution of(R)-3-[N′-t-butoxycarbonyl-N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid methyl ester (20 g, 16 mmol) in HCl/EtOH (1.1 M, 200 mL) wasstirred overnight and then concentrated in vacuo. The residue wasdispersed in EtOAc (2×40 mL), and the precipitate was collected byfiltration to give the title compound as an off-white solid HCl salt(8.8 g). LC-MS: 367 [M+H]⁺. ¹H NMR (300 MHz, DMSO-d₆) δ 1.05 (t, J=7.2Hz, 3 H), 3.05-3.03 (q, J=7.2 Hz, 2 H), 4.06-3.95 (m, 4H), 4.42 (br, 1H), 6.46 (br, 1 H), 7.62-7.40 (m, 7 H), 9.42 (s, 3 H).

Preparation 40(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid 2-oxo-2-phenylethyl Ester

3-[N-(4-Bromobenzyl)hydrazino]-2-hydroxypropionic acid ethyl ester (3.1g, 9.6 mmol) was combined with di-t-butyldicarbonate (4.2 g, 19.2 mmol)and DCM (92.4 mL, 1.4 mol). DIPEA (5.0 mL, 28.8 mmol) was added and theresulting mixture was stirred at room temperature for 24 hours. Themixture was concentrated and the reside was dissolved into DCM andpurified by flash chromatography (10-95% EtOAc in hexanes). The cleanfractions were collected and concentrated to yield Compound 1 as a whitepowder (4.0 g).

Compound 1 (3.5 g, 8.4 mmol) was combined with5-chloro-2-fluorophenylboronic acid (1.8 g, 10.1 mmol) and K₂CO₃ (3.5 g,25.2 mmol) in EtOH (29.4 mL, 503 mmol) and water (7.6 mL, 419 mmol). Theresulting mixture was placed under nitrogen and SilicaCat DPP-Pd(0.28mmol/g loading; 3.0 g, 839 μmol) was then added. The mixture wasmicrowaved at 120° C. for 15 minutes. The mixture was then filtered andevaporated under reduced pressure. The crude residue was purified usingflash chromatography (10-90% EtOAc in hexanes) to yield Compound 2 (2.0g).

Compound 2 (500 mg, 1 mmol) was combined with K₂CO₃ (315 mg, 2.3 mmol)in DMF (5.3 mL, 68.4 mmol). 2-Bromoacetophenone (249 mg, 1.3 mmol) wasthen added and the resulting mixture was stirred at room temperature for15 minutes. The mixture was then purified using flash chromatography(50-100% EtOAc in Hexanes). This purified material (605 mg) was thendissolved in MeCN (3.6 mL, 68.4 mmol). A solution of 4 M HCl in1,4-dioxane (1.4 mL, 5.7 mmol) was then added, and the resulting mixturewas stirred for 1 hour to yield the title compound (245 mg).

Preparation 41(R)-3-[N′-t-Butoxyoxalyl-N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicAcid

t-Butyl oxalyl chloride was prepared by adding oxalyl chloride (102 μL,1.2 mmol) to a solution of t-butyl alcohol (35 μL, 361 μmol) in ether(632 μL, 6.0 mmol). The resulting mixture was stirred at roomtemperature for 15 minutes and then concentrated in vacuo to yield aclear colorless liquid.

(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid 2-oxo-2-phenylethyl ester (55.0 mg, 120 μmol) was dissolved in DCM(463 μL, 7.2 mmol). The t-butyl oxalyl chloride was added, and theresulting mixture was stirred at room temperature for 30 minutes, andthen concentrated in vacuo. The resulting residue was dissolved in AcOH(411 μL, 7.2 mmol). Zinc (394 mg, 6.0 mmol) was added to the mixture,which was then stirred at room temperature for 10 minutes. The mixturewas filtrated and purified (Interchim reverse phase column) to yield thetitle compound (25.0 mg).

Preparation 42(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicAcid 5-methyl-2-oxo-[1,3]dioxol-4-ylmethyl Ester

LiOH hydrate (3 g, 73 mmol) in water (60 mL) was added to(R)-3-[N′-t-butoxycarbonyl-N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid methyl ester (16.5 g, 36.5 mmol) in MeOH (300 mL). The mixture wasstirred at room temperature for 2 hours, and the MeOH was evaporated invacuo. The mixture was adjusted to pH=5 with 1 M aqueous HCl, and theresidue was extracted with EtOAc (2×300 mL). The combined organic layerswere dried over anhydrous Na₂SO₄, and concentrated in vacuo to yieldCompound 1 as a white solid (18 g). LC-MS: 383 [M-tBu+H].

To a solution of Compound 1 (1.5 g, 3.42 mmol), K₂CO₃ (0.95 g, 6.84mmol) and potassium iodide (20 mg) in DMF (40 mL) was added4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (0.8 g, 4.1 mmol) in DMF (15mL). The resulting mixture was stirred for 4 hours at room temperature.Saturated aqueous NaCl (30 mL) was added and the mixture was extractedwith EtOAc (2×50 mL). The combined organic layers were dried overanhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified bycolumn chromatography (hexanes/EtOAc=1:1) to yield Compound 2 as ayellow solid (930 mg). LC-MS: 495 [M-tBu+H]⁺.

Compound 2 (400 mg, 0.73 mmol) was dissolved in MeCN (20 mL), and cooledto 0° C. N-trimethylsilylimidazole (290 mg, 1.46 mmol) was addeddropwise and the resulting mixture was stirred for 2 hours. MeOH (50 mL)was added to quench the reaction. The mixture was washed with saturatedaqueous NaCl (2×50 mL) and extracted with DCM (2×80 mL). The combinedorganic layers were dried over anhydrous Na₂SO₄ and concentrated invacuo. The product was collected to yield the title compound as a yellowsolid (200 mg). LC-MS: 451 [M+H]⁺.

Preparation 43(R)-3-[N′-t-Butoxyoxalyl-N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)-hydrazino]-2-hydroxypropionicAcid

To a mixture of(R)-3-[N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid 2-oxo-2-phenylethyl ester (2.0 g, 4.4 mmol) in DCM (10 mL) wasadded dropwise t-butyl 2-chloro-2-oxoacetate (1.5 g, 8.8 mmol) at 0° C.under nitrogen. DIPEA (1.15 g, 8.8 mmol) was then added dropwise, andthe resulting mixture was stirred for 5 minutes at 0° C. The solvent wasremoved by evaporation and the residue was purified by columnchromatography (PE:EtOAc=2:1) to yield Compound 1 as a yellow liquid(2.0 g). LC-MS: 585[M+H]⁺.

A mixture of Compound 1 (2.0 g, 3.4 mmol) and Zn (15.5 g, 240 mmol) inAcOH (15 mL) was stirred for 1 hour at room temperature, then filtered.Water (30 mL) was added to the filtrate, and the mixture was extractedwith EtOAc (3×40 mL). The combined organic layers were washed withsaturated aqueous NaCl(2×50 mL), dried over anhydrous Na₂SO₄, andconcentrated in vacuo. The residue was purified by column chromatography(DCM/MeOH=10:1) to yield the title compound as a yellow liquid (1.4 g).LC-MS: 467 [M+H]⁺.

Example 1 (R)-5-Biphenyl-4-yl-2-hydroxy-4-(oxalylamino)pentanoic Acid

(R)-4-Amino-5-biphenyl-4-yl-2-hydroxypentanoic acid ethyl ester (HClsalt; 47 mg, 0.2 mmol) and ethyl oxalyl chloride (18.4 nL, 1.1 eq) werecombined with DIPEA (52.2 nL, 0.3 mmol) in DMF (0.3 mL)/DCM (0.3 mL).The mixture was stirred at room temperature until the reaction wascomplete. The solvent was removed and the residue was dissolved in EtOH(750 nL) and 1 M aqueous NaOH (750 nL), and stirred at room temperatureovernight. The solvent was removed and the residue was purified bypreparative HPLC to yield the title compound (28.2 mg, purity 100%). MSm/z [M+H]⁺ calc'd for C₁₉H₁₉NO₆, 358.12; found 358.0.

Example 2 A.(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid

A solution of ethyl oxalyl chloride (70.7 μL, 0.6 mmol) in DIPEA (165μL, 0.9 mmol) was added to a solution of(2R,4R)-4-amino-5-(4-bromophenyl)-2-hydroxypentanoic acid ethyl ester(100 mg, 0.3 mmol) and DCM (0.7 mL), and the resulting mixture wasstirred at room temperature for 10 minutes, followed by evaporation ofthe solvent under reduced pressure. 3-Chlorophenylboronic acid, pinacolester (112 mg, 468 μmol), K₂CO₃ (97 mg, 702 μmol), EtOH (2 mL), andwater (0.6 mL) were added, followed by the addition of SilicaCat® Pd(O)(0.09 mmol/g loading, 260 mg, 23.4 μmol). The mixture was heated at 120°C. for 20 minutes. The reaction mixture was concentrated and 10 M ofaqueous NaOH (316 μL) and THF (4.0 mL) with 1 drop of MeOH was added.The resulting mixture was stirred at room temperature for 1 hour. Theresidue was dissolved in AcOH and purified by preparative HPLC to yieldthe title compound (9 mg, purity 95%). MS m/z [M+H]⁺ calc'd forC₁₉H₁₈ClNO₆, 392.08; found 392.4.

B. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid Ethyl Ester

Oxalyl chloride (54.5 μL, 0.6 mmol) was added to a solution of t-butylalcohol (56.0 μL) in ether (1.0 mL) and the mixture was stirred for 1hour at room temperature. The mixture was concentrated under vacuum anda solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (67.9 mg, 0.2 mmol) and DIPEA (102 μL, 0.6 mmol,) in DCM (1.0 mL)was added to the resulting clear colorless liquid residue. The resultingmixture was stirred at room temperature for 2 hours and concentratedunder vacuum to yield a clear yellow liquid. A 1:1 TFA/DCM (1.6 mL) wasadded to the crude liquid and the reaction mixture was stirred at roomtemperature for 2 hours and concentrated under vacuum to yield a clearyellow liquid. The crude liquid was purified by reverse phasepreparative HPLC (40-90% MeCN/H₂O) to yield the title compound (25.0 mg,purity 95%) as a white solid. MS m/z [M+H]⁺ calc'd for C₂₁H₂₂ClNO₆,420.11; found 420.1.

C.(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-(ethoxyoxalylamino)-2-hydroxypentanoicAcid Ethyl Ester

A solution of ethyl oxalyl chloride (24.6 μL, 0.2 mmol) in DCM (0.4 mL)was added to a solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (69.6 mg, 0.2 mmol) and Et₃N (69.7 μL, 0.5 mmol) in DCM (1.0 mL)at 0° C. over a period of 10 minutes. The resulting mixture was stirredat 0° C. for 30 minutes, and then for 15 minutes at room temperature.Water (2 mL) was added, the layers were separated, and the aqueous layerwas extracted with DCM (2×2 mL). The DCM layers were combined, driedover Na₂SO₄, and concentrated to yield a clear yellow liquid. The crudeliquid was purified by flash chromatography (4 g column, 16 mL/min,using 35% EtOAc/hexanes (2 min), 35-50% (1 min), 50% (4 min), 50-70% (1min) and 70% EtOAc/hexanes (3 min)) to yield the title compound (63.9mg, purity 90%) as a clear colorless liquid which solidified uponstanding to a white solid. MS m/z [M+H]⁺ calc'd for C₂₃H₂₆ClNO₆, 448.14;found 448.2.

D.(2R,4R)-4-(Butoxyoxalylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicAcid Butyl Ester

p-Toluenesulfonic acid monohydrate (849 μg, 4 μmol) was added to asolution of(2R,4R)-5-(3′-chlorobiphenyl-4-yl)-4-(ethoxyoxalylamino)-2-hydroxypentanoicacid ethyl ester (20.0 mg, 45 μmol) in 1-butanol (0.5 mL). The reactionmixture was stirred at 80° C. for 14 hours, at 90° C. for 4 hours, andthen was allowed to cool to room temperature. Saturated aqueous NaHCO₃(2 mL) was added, and the aqueous layer was extracted with DCM (3×2 mL).The DCM layers were combined, dried over Na₂SO₄, and concentrated undervacuum to yield a clear colorless liquid. The crude liquid was purifiedby flash chromatography (4 g column, 40% EtOAc/hexanes) to yield thetitle compound (18.1 mg, purity 99%) as a clear colorless liquid. MS m/z[M+H]⁺ calc'd for C₂₇H₃₄ClNO₆, 504.21; found 504.2.

E.(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(5-methyl-2-oxo-[1,3]dioxol-4-ylmethoxyoxalyl)amino]pentanoicAcid 5-methyl-2-oxo-[1,3]dioxol-4-ylmethyl Ester

Oxalyl chloride (22.4 μL, 264 μmol) was added to a solution of4-hydroxymethyl-5-methyl-[1,3]dioxol-2-one (29.1 mg, 224 μmol) in ether(1.5 mL) and the mixture was stirred at room temperature for 2 hours.The ether was removed under vacuum and the residue was dissolved in DMF(1.5 mL). The resulting solution was added to a solution of(2R,4R)-4-amino-5-(3′-chloro-biphenyl-4-yl)-2-hydroxy-pentanoic acid(65.0 mg, 203 μmol) and NaHCO₃ (51.2 mg) at 0° C. The resulting mixturewas stirred at room temperature for 3 hours, then concentrated undervacuum. The residue was then purified by reverse phase preparative HPLC(30%-90% MeCN/H₂O) to yield compound 1 (19.1 mg) as a white solid.

1-Hydroxybenzotriazole (7.7 mg, 56.8 μmol) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (10.9 mg,56.8 μmol) were added to a solution of compound 1 (19.1 mg, 37.9 μmol)in DCM (1.0 mL) and the mixture was stirred at room temperature for 10minutes. 4-Hydroxymethyl-5-methyl-[1,3]dioxol-2-one (14.8 mg, 114 μmol)and 4-methylmorpholine (7.7 mg, 75.8 μmol) were added and the resultingmixture was stirred at room temperature for 6 hours. Water was added andthe mixture was extracted with DCM (3×1.5 mL). The DCM layers werecombined, dried over Na₂SO₄, and concentrated to yield a yellow liquid.The crude liquid was purified by reverse phase preparative HPLC to yieldthe title compound as a white solid (5.1 mg). MS m/z [M+H]⁺ calc'd forC₂₉H₂₆ClNO₁₂, 616.11; found 616.1.

F.(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-(ethoxyoxalylamino)-2-hydroxypentanoicAcid

Ethyl oxalyl chloride (46.1 μL, 0.4 mmol) was added to a solution of(2R,4R)-4-amino-5-(3′-chloro-biphenyl-4-yl)-2-hydroxy-pentanoic acid(120 mg, 0.4 mmol) and Et₃N (157 μL, 1.1 mmol) in DMF (2.0 mL, 25.8mmol) at 0° C., and the resulting mixture was stirred at roomtemperature for 20 minutes. Additional ethyl oxalyl chloride (30 μL) wasadded and the mixture was stirred an additional 10 minutes. Water (2 mL)was added and the mixture was extracted with DCM (3×2 mL). The extractswere combined, dried over Na₂SO₄ and concentrated to yield a yellowliquid. The crude liquid was purified by (C-18 column chromatography, 20g; 40-90% MeCN in water with 0.05% TFA) to yield the title compound(28.5 mg) as a white solid. MS m/z [M+H]⁺ calc'd for C₂₁H₂₂ClNO₆,420.11; found 420.2.

G.(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(isopropoxyoxalylamino)pentanoicAcid

Chloro-oxo-acetic acid isopropyl ester (62.1 mg, 413 μmol; ˜53 μL) wasadded dropwise to a solution of(2R,4R)-4-amino-5-(3′-chloro-biphenyl-4-yl)-2-hydroxy-pentanoic acid(100 mg, 313 μmol) and Et₃N (157 μL, 1.1 mmol) in DMF (2.0 mL, 25.8mmol) at 0° C., and the resulting mixture was stirred at roomtemperature for 10 minutes. Additional ethyl oxalyl chloride (50 μL) wasadded and the mixture was stirred an additional 10 minutes. Saturatedaqueous NaHCO₃ (5 mL) was added and the mixture was stirred at roomtemperature for 1 hour. The mixture was extracted with DCM (3×3 mL), theextracts were combined, dried over Na₂SO₄, and concentrated to yield ayellow liquid. The crude liquid was purified (pre HPLC C-18 columnchromatography, small column, using 40-95% MeCN in water with 0.05% TFA)to yield the title compound (53.0 mg) as a white solid. MS m/z [M+H]⁺calc'd for C₂₂H₂₄ClNO₆, 434.13; found 434.1.

H.(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(isobutoxyoxalylamino)pentanoicAcid

1.0 M of aqueous HCL (2.5 mL, 2.5 mmol) was added to(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (100 mg, 287 μmol) and the resulting mixture was stirred at 100°C. for 1 hour. The mixture was concentrated to yield(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-pentanoic acid.

Chloro-oxo-acetic acid isobutyl ester (99.4 mg, 604 μmol) was addeddropwise to a solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-pentanoic acid andEt₃N (160 μL, 1.2 mmol) in DMF (2.0 mL, 25.8 mmol) at 0° C., and stirredroom temperature for 10 minutes. Saturated aqueous NaHCO₃ (5 mL) wasadded and the mixture was stirred at room temperature for 2 hours. Themixture was extracted with DCM (3×5 mL), the DCM extracts were combined,washed with saturated aqueous NaCl, dried over Na₂SO₄, and concentratedto yield a white solid residue. The crude solid was purified bepreparative HPLC C18 column chromatography (small column; 40-90% MeCN inwater with 0.05% TFA) to yield the title compound (40.0 mg) as a whitesolid. MS m/z [M+H]⁺ calc'd for C₂₃H₂₆ClNO₆, 448.14; found 448.1.

I. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid Isobutyl Ester

4.0 M HCl in 1,4-dioxane (216 μL, 862 μmol) was added to a suspension of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (75.0 mg, 216 μmol) in isobutyl alcohol (0.5 mL, 5.4 mmol), andthe resulting mixture was stirred at 60° C. for 2 hours. The mixture wasthen concentrated in vacuo to yield a white solid. The white solid wasdissolved in DCM and DIPEA (113 μL, 647 μmol) was then added to themixture followed by 0.22 mL of a 1M t-butyl oxalyl chloride solution inDCM (0.2 mmol) dropwise. The resulting mixture was stirred at roomtemperature for 30 minutes and then concentrated in vacuo to yield ayellow liquid. A TFA/DCM (1:1, 1.3 mL, 7.7 mmol) solution was added tothe yellow liquid and the resulting mixture was stirred at roomtemperature for 30 minutes and then concentrated in vacuo to yield aclear yellow liquid. The crude liquid was purified (preparative scaleHPLC C18 column chromatography, 40-90% MeCN in water with 0.05% TFA) toyield the title compound (70.5 mg, purity 99%) as a white solid. MS m/z[M+H]⁺ calc'd for C₂₃H₂₆ClNO₆, 448.14; found 448.1.

J. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid Isopropyl Ester

4.0 M HCl in 1,4-dioxane (216 μL, 862 μmol) was added to a suspension of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (75.0 mg, 216 μmol) in isopropyl alcohol (0.5 mL, 6.5 mmol) andthe resulting mixture was stirred at 60° C. overnight. The mixture wasthen concentrated in vacuo to yield a white solid. The white solid wasdissolved in DCM and DIPEA (113 μL, 647 μmol) was then added to themixture followed by 0.22 mL of a 1M t-butyl oxalyl chloride solution inDCM (0.2 mmol) dropwise. The resulting mixture was stirred at roomtemperature for 30 minutes and then concentrated in vacuo to yield ayellow liquid. A TFA/DCM (1:1, 1.3 mL, 7.7 mmol) solution was added tothe yellow liquid and the resulting mixture was stirred at roomtemperature for 30 minutes and then concentrated in vacuo to yield aclear yellow liquid. The crude liquid was purified (preparative scaleHPLC C18 column chromatography, 40-90% MeCN in water with 0.05% TFA) toyield the title compound (62.8 mg, purity 98%) as a white solid. MS m/z[M+H]⁺ calc'd for C₂₂H₂₄ClNO₆, 434.13; found 434.1.

K. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid Heptyl Ester

4.0 M HCl in 1,4-dioxane (216 μL, 862 μmol) was added to a suspension of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (75.0 mg, 216 μmol) in 1-heptanol (250 μL, 1.8 mmol) and theresulting mixture was stirred at 60° C. for 2 hours. The mixture wasthen concentrated in vacuo to yield a white solid, which was purified(Interchim reverse phase chromatography column; 30-90% MeCN in watergradient with 0.5% TFA). The purified white solid was dissolved in DCMand DIPEA (113 μL, 647 μmol) was then added to the mixture followed by0.22 mL of a 1M t-butyl oxalyl chloride solution in DCM (0.2 mmol)dropwise. The resulting mixture was stirred at room temperature for 30minutes and then concentrated in vacuo to yield a yellow liquid. ATFA/DCM (1:1, 1.3 mL, 7.7 mmol) solution was added to the yellow liquidand the resulting mixture was stirred at room temperature for 30 minutesand then concentrated in vacuo to yield a clear yellow liquid. The crudeliquid was purified (preparative scale HPLC C18 column chromatography,40-90% MeCN in water with 0.05% TFA) to yield the title compound (43.3mg, purity 99%) as a white solid. MS m/z [M+H]⁺ calc'd for C₂₆H₃₂ClNO₆,490.19; found 490.2.

L. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid 3,3,3-Trifluoropropyl Ester

4.0 M HCl in 1,4-dioxane (287 μL, 1.2 mmol) was added to a suspension of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (50.0 mg, 144 μmol) in 3,3,3-trifluoropropan-1-ol (492 mg, 4.3mmol) and the resulting mixture was stirred at 80° C. for 12 hours. Themixture was then concentrated in vacuo to yield a white solid, which wasdissolved in DCM (1.0 mL) and 0.2 mL of a 1M t-butyl oxalyl chloridesolution in DCM (0.2 mmol). DIPEA (75.1 μL, 431 μmol) was then addeddropwise and the resulting mixture was stirred at room temperature for30 minutes and then concentrated in vacuo to yield a yellow liquid. ATFA/DCM (1:1, 1.3 mL, 7.7 mmol) solution was added to the yellow liquidand the resulting mixture was stirred at room temperature for 30 minutesand then concentrated in vacuo to yield a clear yellow liquid. The crudeliquid was purified (preparative scale HPLC C18 column chromatography,40-90% MeCN in water with 0.05% TFA) to yield the title compound (44.9mg, purity 99%) as white solid. MS m/z [M+H]⁺ calc'd for C₂₂H₂₁ClF₃NO₆,488.10; found 488.1.

M. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid 2,2,2-Trifluoroethyl Ester

4.0 M HCl in 1,4-dioxane (287 μL, 1.2 mmol) was added to a suspension of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (50.0 mg, 144 μmol) in 2,2,2-trifluoroethanol (0.5 mL, 6.9 mmol)and the resulting mixture was stirred at 110° C. for 12 hours. Themixture was then concentrated in vacuo to yield a white solid, which wasdissolved in DCM (1.0 mL) and 0.2 mL of a 1M t-butyl oxalyl chloridesolution in DCM (0.2 mmol). DIPEA (75.1 μL, 431 μmol) was then addeddropwise and the resulting mixture was stirred at room temperature for30 minutes and then concentrated in vacuo to yield a yellow liquid. ATFA/DCM (1:1, 1.3 mL, 7.7 mmol) solution was added to the yellow liquidand the resulting mixture was stirred at room temperature for 30 minutesand then concentrated in vacuo to yield a clear yellow liquid. The crudeliquid was purified (preparative scale HPLC C18 column chromatography,40-90% MeCN in water with 0.05% TFA) to yield the title compound (22.5mg, purity 98%) as a white solid. MS m/z [M+H]⁺ calc'd forC₂₁H₁₉ClF₃NO₆, 474.09; found 474.1.

N.(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(3,3,3-trifluoropropoxyoxalyl)-amino]pentanoicAcid

Benzyl alcohol (13.0 mL, 126 mmol) was added to(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (1.9 g, 5.3 mmol) followed by 4.0 M HCl in 1,4-dioxane (5.3 mL,21.3 mmol), and the mixture was stirred at 60° C. for 1 hour. Themixture was purified (Interchim reverse phase chromatography column;30-90% MeCN in water with 0.05% TFA) to yield(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxy-pentanoic acidbenzyl ester (2.2 g) as a white solid. (evaporated in vacuo with water(4×300 mL) to remove excess benzyl alcohol).

3,3,3-trifluoropropyl oxalyl chloride was prepared by adding oxalylchloride (51.6 μL, 610 μmol) to a solution of 3,3,3-trifluoropropan-1-ol(62.6 mg, 549 μmol) in ether (500 μL, 4.8 mmol). The resulting mixturewas stirred at room temperature for 1 hour and then concentrated invacuo yield a clear colorless liquid. A ˜1M solution of the oxalylchloride was prepared by dissolving the resulting liquid in ˜0.61 mLDCM.

A 3,3,3-trifluoropropyl oxalyl chloride solution (˜140 μL) was added toa solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acidbenzyl ester (50.0 mg, 122 μmol) in DCM (1.0 mL) at 0° C., and themixture was stirred at 0° C. for 15 minutes. Saturated aqueous NaHCO₃ (1mL) was added and the mixture was stirred at room temperature for 1hour. The layers were separated and the aqueous layer was extracted withDCM (2×2 mL). The DCM layers were combined, dried over Na₂SO₄, andconcentrated to yield a clear yellow liquid. 10% Pd/C, 50% wet (0.45mmol/g loading; 13.6 mg, 6.1 μmol) was added to a solution of the yellowliquid in DCM and THF (1.0 mL), and the mixture was stirred underhydrogen for 30 minutes. The mixture was filtered and the filtrate wasconcentrated to yield a clear yellow liquid. The crude liquid waspurified by preparative HPLC (40-90% MeCN in water with 0.05% TFA) toyield the title compound (16.5 mg, purity 99%) as a white solid. MS m/z[M+H]⁺ calc'd for C₂₂H₂₁ClF₃NO₆, 488.10; found 4880.

O. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid 2,2,3,3,3-Pentafluoropropyl Ester

A ˜1M solution of t-butyl oxalyl chloride (˜0.2 mL) was added to asolution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid2,2,3,3,3-pentafluoropropyl ester (50.0 mg, 111 μmol) in DCM (1.0 mL) at0° C. followed by the dropwise addition over 10 minutes of DIPEA (21.2nL, 122 nmol). The mixture was stirred at 0° C. for 15 minutes.Saturated aqueous NaHCO₃ (5 mL) was added and the mixture was extractedwith DCM (3×5 mL). The DCM extracts were combined, dried over Na₂SO₄,and concentrated to yield a clear colorless liquid. The crude liquid waspurified by flash chromatography (50% EtOAc/hexanes to yield a clearcolorless liquid. 1:1 TFA/DCM (1.0 mL) was added to a solution of thecolorless liquid and stirred at room temperature for 30 minutes. Themixture was concentrated in vacuo to yield a clear yellow liquid. Thecrude liquid was purified preparative HPLC (40%-90% MeCN in water with0.05% TFA) to yield the title compound (21.6 mg, purity 98%) as a whitesolid. MS m/z [M+H]⁺ calc'd for C₂₂H₁₉ClF₅NO₆, 524.08; found 524.0.

P. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid 5-Methyl-2-oxo-[1,3]dioxol-4-ylmethyl Ester

A ˜1M solution of t-butyl oxalyl chloride (160 nL) was added to asolution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid5-methyl-2-oxo[1,3]dioxol-4-ylmethyl ester (50.0 mg, 116 nmol) in DCM(1.00 mL, 15.6 mmol) at 0° C. followed by the dropwise addition over 10minutes of N,N-diisopropylamine (17.8 μL, 127 mmol). The resultingmixture was stirred at 0° C. for 15 minutes, then concentrated in vacuo.1:1 TFA/DCM (1.0 mL, 6.2 mmol) was added to the residue and theresulting mixture was stirred at room temperature for 30 minutes. Themixture was concentrated in vacuo to yield a clear yellow liquid. Thecrude liquid was purified (preparative scale C18 column chromatography,small column, using 30-90% MeCN in water with 0.05% TFA) to yield thetitle compound as a white solid (9.6 mg). MS m/z [M+H]⁺ calc'd forC₂₄H₂₂ClNO₉, 504.10; found 504.0.

Q. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid Butyryloxymethyl Ester

A ˜1M solution of t-butyl oxalyl chloride (160 μL) was added to asolution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acidbutyryloxymethyl ester (48.6 mg, 116 μmol) in DCM (1.00 mL, 15.6 mmol)at 0° C. followed by the dropwise addition over 10 minutes ofN,N-diisopropylamine (17.8 μL, 127 mmol). The resulting mixture wasstirred at 0° C. for 15 minutes, then concentrated in vacuo. 1:1 TFA/DCM(1.0 mL, 6.2 mmol) was added to the residue and the resulting mixturewas stirred at room temperature for 30 minutes. The mixture wasconcentrated in vacuo to yield a clear yellow liquid. The crude liquidwas purified (preparative scale C18 column chromatography, small column,using 30-90% MeCN in water with 0.05% TFA) to yield the title compoundas a white solid (10.2 mg). MS m/z [M+1-1]⁺ calc'd for C₂₄H₂₆ClNO₈,492.13; found 492.0.

R.(2R,4R)-5-(3′-Chloro-biphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid Acetoxymethyl Ester

To a solution of(2R,4R)-4-(t-butoxyoxalylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (200 mg, 450 μmol) and bromomethyl acetate (97 mg, 0.9 mmol) in DMF(2 mL) was added 2, 6-lutidine (144 mg, 1.3 mmol) and NaI (67 mg, 450μmol). After stirring at room temperature for 24 hours, the mixture wasdiluted with water (20 mL) and extracted with EtOAc (2×20 mL). Thecombined organic layers were washed with saturated aqueous NaCl (2×70mL), dried over anhydrous Na₂SO₄, filtered, and concentrated to give thecrude product which was further purified by preparative TLC(PE:EtOAc=2:1) to yield Compound 1 (100 mg) as a yellow solid. LC-MS:542[M+Na]⁺.

To a solution of Compound 1 (100 mg, 0.2 mmol) in DCM (5 mL) was addedTFA (2 mL) at 0° C. The mixture was stirred at room temperature for 2hours, the solvent was removed, and the residue was further purified bypreparative TLC (DCM:MeOH=8:1) to yield the title compound as a whitesolid (10 mg). LC-MS: 464[M+H]⁺. ₁H NMR (400 MHz, MeOD) δ 7.61 (s, 1H),7.55 (d, J=8.0 Hz, 3H), 7.42 (t, J=7.8 Hz, 1H), 7.34 (d, J=8.1 Hz, 3H),5.78 (s, 2H), 4.40 (s, 1H), 4.31 (t, J=5.9 Hz, 1H), 2.94 (ddd, J=22.0,13.8, 7.2 Hz, 2H), 2.09 (m, 5H).

S. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid Ethoxycarbonyloxymethyl Ester

To a solution of(2R,4R)-4-(t-butoxyoxalylamino)-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoicacid (100 mg, 220 μmol) and chloromethyl ethyl carbonate (61 mg, 440μmol) in DMF (3 mL) was added 2, 6-lutidine (72 mg, 660 μmol) and NaI(33 mg, 220 mmol). After stirring at room temperature for 24 hours, themixture was diluted with water (20 mL) and extracted with EtOAc (2×20mL). The combined organic layers were washed with saturated aqueous NaCl(2×70 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated togive the crude product which was further purified by preparative TLC(PE:EtOAc=2:1) to yield Compound 1 (40 mg) as a yellow solid. LC-MS:572[M+Na]⁺.

To a solution of Compound 1 (40 mg, 70 μmol) in DCM (3 mL) was added TFA(1 mL) at 0° C. The mixture was stirred at room temperature for 2 hours,the solvent was removed, and the residue was further purified bypreparative TLC (DCM:MeOH=8:1) to yield the title compound as a whitesolid (18 mg). LC-MS: 494[M+H]⁺. ¹H NMR (400 MHz, MeOD) δ 7.55 (m, 4H),7.38 (m, 4H), 5.80 (d, J=18.6 Hz, 2H), 4.34 (s, 2H), 4.21 (dd, J=14.3,7.1 Hz, 2H), 2.95 (m, 2H), 2.07 (d, J=28.0 Hz, 2H), 1.29 (dd, J=12.6,5.5 Hz, 3H). MS m/z [M+H]⁺ calc'd for C₂₃H₂₄ClNO₉, 494.11; found 494.

T.(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(2-methoxyethoxyoxalyl)amino]-pentanoicAcid

DIPEA (64 μL, 366 μmol) was added to a solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acidbenzyl ester (50.0 mg, 122 μmol) in DCM (3 mL) followed by the dropwiseaddition of a 1.0M chloro-oxo-acetic acid 2-methoxyethyl ester (22 mg,134 μmol) solution in DCM. The resulting mixture was stirred at roomtemperature for 30 minutes, then concentrated to yield a clear yellowliquid. The crude liquid was purified (Interchim C18 chromatographycolumn, 20 g, 340-90% MeCN in water with 0.05% TFA). THF (3 mL) wasadded to the purified material, followed by the addition of palladiumcarbon (10 wt % on carbon, wet 50 g, 12.9 mg, 12 μmol) and the mixturewas stirred under hydrogen for 30 minutes. The mixture was filtered andconcentrated in vacuo, and the residue dissolved in AcOH (0.5 mL) andpurified by preparative HPLC to yield the title compound (9.8 mg). MSm/z [M+H]⁺ calc'd for C₂₂H₂₄ClNO₇, 450.12; found 450.2.

U.(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-[(2-phenoxyethoxyoxalyl)amino]-pentanoicAcid

DIPEA (64 μL, 366 μmol) was added to a solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acidbenzyl ester (50.0 mg, 122 μmol) in DCM (3 mL) followed by the dropwiseaddition of a 1.0M chloro-oxo-acetic acid 2-phenoxyethyl ester (31 mg,134 μmol) solution in DCM. The resulting mixture was stirred at roomtemperature for 30 minutes, then concentrated to yield a clear yellowliquid. The crude liquid was purified (Interchim C18 chromatographycolumn, 20 g, 340-90% MeCN in water with 0.05% TFA). THF (3 mL) wasadded to the purified material, followed by the addition of palladiumcarbon (10 wt % on carbon, wet 50 g, 12.9 mg, 12 μmol) and the mixturewas stirred under hydrogen for 30 minutes. The mixture was filtered andconcentrated in vacuo, and the residue dissolved in AcOH (0.5 mL) andpurified by preparative HPLC to yield the title compound (3.5 mg). MSm/z [M+H]⁺ calc'd for C₂₇H₂₆ClNO₇, 512.14; found 512.2.

V.(2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-4-[(3-ethoxypropoxyoxalyl)amino]-2-hydroxy-pentanoicAcid

DIPEA (64 μL, 366 μmol) was added to a solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acidbenzyl ester (50.0 mg, 122 μmol) in DCM (3 mL) followed by the dropwiseaddition of a 1.0M chloro-oxo-acetic acid 3-ethoxypropyl ester (26 g,134 μmol) solution in DCM. The resulting mixture was stirred at roomtemperature for 30 minutes, then concentrated to yield a clear yellowliquid. The crude liquid was purified (Interchim C18 chromatographycolumn, 20 g, 340-90% MeCN in water with 0.05% TFA). THF (3 mL) wasadded to the purified material, followed by the addition of palladiumcarbon (10 wt % on carbon, wet 50 g, 12.9 mg, 12 μmol) and the mixturewas stirred under hydrogen for 30 minutes. The mixture was filtered andconcentrated in vacuo, and the residue dissolved in AcOH (0.5 mL) andpurified by preparative HPLC to yield the title compound (10.5 mg). MSm/z [M+H]⁺ calc'd for C₂₄H₂₈ClNO₇, 478.16; found 478.2.

W. (2R,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid (S)-2-Methoxycarbonylamino-3-methylbutyryloxymethyl Ester

Following the methods described herein, the title compound was alsoprepared (12.6 mg). MS m/z [M+H]⁺ calc'd for C₂₇H₃₁ClN₂O₁₀, 579.17;found 579.2.

Example 3 A.(2R,4R)-5-(2′,5′-Dichlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid

A solution of ethyl oxalyl chloride (42.4 μL, 0.4 mmol) in DCM (0.4 mL,6 mmol) was added to a solution of(2R,4R)-4-amino-5-(4-bromophenyl)-2-hydroxypentanoic acid ethyl ester(80 mg, 0.2 mmol) and Et₃N (0.1 mL, 0.8 mmol) in DCM (1 mL), and theresulting mixture was stirred at room temperature for 30 minutes, thenevaporated under reduced pressure. The product was then combined with2,5-dichlorophenylboronic acid (72.4 mg, 0.4 mmol), K₂CO₃ (104.9 mg, 759μmol), EtOH (0.9 mL), and water (0.2 mL). The mixture was placed undernitrogen and SilicaCat®DPP-Pd (0.28 mmol/g loading, 90.4 mg, 25.3 μmol)was added. The mixture was microwaved at 120° C. for 20 minutes, thenfiltered. 1 M Aqueous LiOH (2.5 mL, 2.5 mmol) was added to yield thetitle compound (11.9 mg, purity 100%). MS m/z [M+H]⁺ calc'd forC₁₉H₁₇Cl₂NO₆, 426.04; found 426.0.

B.(2R,4R)-5-(2′,5′-Dichlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid Isobutyl Ester

4.0 M HCl in 1,4-dioxane (196 μL, 785 μmol) was added to a suspension of(2R,4R)-4-amino-5-(2′,5′-dichlorobiphenyl-4-yl)-2-hydroxy-pentanoic acidethyl ester (75.0 mg, 196 μmol) in isobutyl alcohol (0.5 mL, 5.4 mmol),and the resulting mixture was stirred at 60° C. for 2 hours. The mixturewas then concentrated in vacuo to yield a white solid. The white solidwas dissolved in DCM (1 mL) and DIPEA (102 μL, 588 μmol) was then addedto the mixture followed by 0.2 mL of a 1M t-butyl oxalyl chloridesolution in DCM (0.2 mmol) dropwise. The resulting mixture was stirredat room temperature for 30 minutes and then concentrated in vacuo toyield a yellow liquid. A TFA/DCM (1:1, 1.1 mL, 7.0 mmol) solution wasadded to the yellow liquid and the resulting mixture was stirred at roomtemperature for 30 minutes and then concentrated in vacuo to yield aclear yellow liquid. The crude liquid was purified (preparative scaleHPLC C18 column chromatography, 40-90% MeCN in water with 0.05% TFA) toyield the title compound (80 mg, purity 99% as a white solid. MS m/z[M+H]⁺ calc'd for C₂₃H₂₅Cl₂NO₆, 482.11; found 482.1.

C.(2R,4R)-5-(2′,5′-Dichlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid Isopropyl Ester

4.0 M HCl in 1,4-dioxane (196 μL, 785 μmol) was added to a suspension of(2R,4R)-4-amino-5-(2′,5′-dichlorobiphenyl-4-yl)-2-hydroxy-pentanoic acidethyl ester (75.0 mg, 196 μmol) in isopropyl alcohol (0.5 mL, 6.5 mmol),and the resulting mixture was stirred at 60° C. overnight. The mixturewas then concentrated in vacuo to yield a white solid. The white solidwas dissolved in DCM (1 mL) and DIPEA (102 nL, 588 nmol) was then addedto the mixture followed by 0.2 mL of a 1M t-butyl oxalyl chloridesolution in DCM (0.2 mmol) dropwise. The resulting mixture was stirredat room temperature for 30 minutes and then concentrated in vacuo toyield a yellow liquid. A TFA/DCM (1:1, 1.1 mL, 7.0 mmol) solution wasadded to the yellow liquid and the resulting mixture was stirred at roomtemperature for 30 minutes and then concentrated in vacuo to yield aclear yellow liquid. The crude liquid was purified (preparative scaleHPLC C18 column chromatography, 40-90% MeCN in water with 0.05% TFA) toyield the title compound (60.6 mg, purity 98%) as a white solid. MS m/z[M+H]⁺ calc'd for C₂₂H₂₃Cl₂NO₆, 468.09; found 468.1.

D.(2R,4R)-5-(2′,5′-Dichlorobiphenyl-4-yl)-2-hydroxy-4-(isobutoxyoxalylamino)-pentanoicAcid

1.0 M Aqueous HCl (3.5 mL, 3.5 mmol) was added to(2R,4R)-4-amino-5-(2′,5′-dichlorobiphenyl-4-yl)-2-hydroxy-pentanoic acidethyl ester (155 mg, 405 nmol) and the mixture was stirred at 100° C.for 1 hour then concentrated. The product was combined with Et₃N (226nL, 1.6 mmol) in DMF (2.5 mL, 32.3 mmol). Chloro-oxo-acetic acidisobutyl ester (140 mg, 851 nmol) was added dropwise at 0° C. and theresulting mixture was stirred at room temperature for 10 minutes.Saturated aqueous NaHCO₃ (5 mL) was added and the mixture was stirred atroom temperature for 2 hours. The mixture was extracted with DCM (3×5mL), the extracts were combined, washed with a saturated aqueous NaClsolution, dried over Na₂SO₄ and concentrated to yield a white solidresidue. The crude solid was purified via preparative HPLC (C18 column;40-90% MeCN in water with 0.05% TFA) to yield the title compound (98.0mg, purity 99%) as a white solid. MS m/z [M+H]⁺ calc'd for C₂₃H₂₅Cl₂NO₆,482.11; found 482.1.

Example 4(2R,4R)-5-(3-Chlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoicAcid

A solution of ethyl oxalyl chloride (41 μL, 0.4 mmol) in DCM (0.5 mL)was added to a solution of(3R,5R)-5-amino-6-(4-bromo-2-chloro-phenyl)-2-ethoxy-hex-1-en-3-ol (96mg, 0.3 mmol) and Et₃N (0.1192 mL, 0.8556 mmol) in DCM (1.4 mL), andstirred for 20 minutes at room temperature. The mixture was evaporatedunder reduced pressure and combined with phenylboronic acid (52.2 mg,0.4 mmol), K₂CO₃ (100 mg, 0.9 mmol), water (0.2 mL), and EtOH (1 mL).The resulting mixture was placed under nitrogen, and SilicaCat®DPP-Pd(0.28 mmol/g loading; 100 mg, 0.03 mmol) was added. The mixture washeated at 120° C. for 20 minutes until the reaction was complete. Themixture was filtered and a solution of 1 M aqueous LiOH (3 mL, 3 mmol)was added. The product was then purified (Interchim reverse phasechromatography column) to yield the title compound (12.6 mg). MS m/z[M+H]⁺ calc'd for C₁₉H₁₈ClNO₆, 392.08; found 392.2.

Example 5

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, thefollowing compounds were prepared:

MS m/z: [M + H]⁺ Ex. R¹ a R⁵ b R⁶ Formula calcd found 1 H 0 — 2 2′-CH₃,5′-Cl C₂₀H₂₀ClNO₆ 406.10 406.0 2 —CH—(CH₃)₂ 0 — 2 2′-CH₃, 5′-ClC₂₃H₂₆ClNO₆ 448.14 448.0 3 —CH₂CH₃ 0 — 2 2′-CH₃, 5′-Cl C₂₂H₂₄ClNO₆434.13 434.4 4 —CH₂—CH—(CH₃)₂ 0 — 2 2′-CH₃, 5′-Cl C₂₄H₂₈ClNO₆ 462.16462.0 5 H 0 — 2 2′-F, 5′-Cl C₁₉H₁₇ClFNO₆ 410.07 410.0 6 —CH₂CH₃ 0 — 22′-F, 5′-Cl C₂₁H₂₁ClFNO₆ 438.10 438.0 7 —CH₂CH—(CH₃)₂ 0 — 2 2′-F, 5′-ClC₂₃H₂₅ClFNO₆ 466.14 466.0 8 —CH—(CH₃)₂ 0 — 2 2′-F, 5′-Cl C₂₂H₂₃ClFNO₆452.12 452.0 9 H 1 3-Cl 1 3′-Cl C₁₉H₁₇Cl₂NO₆ 426.04 426.0 10 —CH₂CH₃ 13-Cl 1 3′-Cl C₂₁H₂₁Cl₂NO₆ 454.07 454.0 11 —CH₂CH—(CH₃)₂ 1 3-Cl 1 3′-ClC₂₃H₂₅Cl₂NO₆ 482.11 482.0 12 —CH—(CH₃)₂ 1 3-Cl 1 3′-Cl C₂₂H₂₃Cl₂NO₆468.09 468.1 13 H 1 3-Cl 2 2′-F, 5′-Cl C₁₈H₁₅Cl₂FN₂O₆ 445.03 445.0

-   1.    (2R,4R)-5-(5′-Chloro-2′-methylbiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)-pentanoic    acid-   2.    (2R,4R)-5-(5′-Chloro-2′-methylbiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)-pentanoic    acid isopropyl ester-   3.    (2R,4R)-5-(5′-Chloro-2′-methylbiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoic    acid ethyl ester-   4.    (2R,4R)-5-(5′-Chloro-2′-methylbiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoic    acid isobutyl ester-   5.    (2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoic    acid-   6.    (2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoic    acid ethyl ester-   7.    (2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoic    acid isobutyl ester-   8.    (2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoic    acid isopropyl ester-   9.    (2R,4R)-5-(3,3′-Dichlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoic    acid-   10.    (2R,4R)-5-(3,3′-Dichlorobiphenyl-4-yl)-2-hydroxy-4-(isobutoxyoxalylamino)-pentanoic    acid-   11.    (2R,4R)-5-(3,3′-Dichlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoic    acid isobutyl ester-   12.    (2R,4R)-5-(3,3′-Dichlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoic    acid isopropyl ester-   13.    (R)-3-[N-(3,5′-Dichloro-2′-fluorobiphenyl-4-ylmethyl)-N′-oxalyl-hydrazino]-2-hydroxypropionic    acid

MS m/z: [M + H]⁺ Ex. R⁴ a R⁵ b R⁶ Formula calcd found 14 —CH₂CH₃ 0 — 22′-F, 5′-Cl C₂₁H₂₁ClFNO₆ 438.10 438.2 15 —CH—(CH₃)₂ 0 — 2 2′-F, 5′-ClC₂₂H₂₃ClFNO₆ 452.12 452.2 16 —CH₂CH—(CH₃)₂ 0 — 2 2′-F, 5′-ClC₂₃H₂₅ClFNO₆ 466.14 466.4 17 —CH₂CH—(CH₃)₂ 1 3-Cl 1 3′-Cl C₂₃H₂₅Cl₂NO₆482.11 482.1

-   14.    (2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-4-(ethoxyoxalylamino)-2-hydroxy-pentanoic    acid-   15.    (2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-(isopropoxyoxalyl-amino)-pentanoic    acid-   16.    (2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxy-4-(isobutoxyoxalylamino)-pentanoic    acid-   17.    (2R,4R)-5-(3,3′-Dichlorobiphenyl-4-yl)-2-hydroxy-4-(oxalylamino)pentanoic    acid ethyl ester

Example 6 A.(2S,4S)-5-Biphenyl-4-yl-2-hydroxymethyl-4-(oxalylamino)pentanoic Acid

Ethyl oxalyl chloride (27 μL, 0.2 mmol, 1.1 eq) was added to a solutionof (2S,4S)-4-amino-5-biphenyl-4-yl-2-hydroxymethyl-pentanoic acid ethylester (HCl salt; 80 mg, 0.22 mmol) in DMF (0.5 mL)/DCM (0.5 mL), andstirred at room temperature for 20 minutes. The solvent was removed andthe residue was dissolved in LiOH (monohydrate; 92.2 mg, 2.2 mmol),water (1.0 mL) and EtOH (2.0 mL), and stirred at room temperature for 30minutes. The reaction was quenched with AcOH and the solvent wasremoved. The residue was dissolved into AcOH/MeCN and purified bypreparative HPLC. The clean fractions were combined and lyophilized toyield the title compound (37 mg, purity 95%). MS m/z [M+H]⁺ calc'd forC₂₀H₂₁NO₆, 372.14; found 372.2.

B. (2S,4S)-5-Biphenyl-4-yl-2-hydroxymethyl-4-(oxalylamino)pentanoic AcidEthyl Ester

Oxalyl chloride (232 μL, 2.8 mmol) and t-butyl alcohol (228 μL) werecombined in ether (6.7 mL) under nitrogen at 0° C. The resulting mixturewas stirred for 30 minutes at room temperature. The solvent wasevaporated under vacuum to form chloro-oxo-acetic acid t-butyl ester,which was then dissolved in DCM (10 mL) and combined with(2S,4S)-4-amino-5-biphenyl-4-yl-2-hydroxymethyl-pentanoic acid ethylester (HCl salt; 667 mg, 1.8 mmol), which had been dissolved in DCM withEt₃N (2.6 mL) at 0° C. The resulting mixture was stirred for 5 minutesat room temperature. The crude product was concentrated, dissolved inDCM and purified by flash chromatography (20-80% EtOAc/hexanes). Thesolvent was removed and the residue was dissolved in DCM (5 mL) and TFA(1 mL), and stirred for 1 hour. The product was dried under vacuum andpurified by preparative HPLC to yield the title compound (135 mg, purity95%). MS m/z [M+H]⁺ calc'd for C₂₂H₂₅NO₆, 400.17; found 400.2.

Example 7 A.(2S,4S)-5-(2′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-4-(oxalylamino)pentanoicAcid Butyl Ester

Oxalyl chloride (44.1 μL. 0.5 mmol) and t-butyl alcohol (46.5 μL) werecombined in ether (1 mL) and was stirred for 30 minutes at roomtemperature. The solvent was evaporated under vacuum to formchloro-oxo-acetic acid t-butyl ester, which was then dissolved in DCM (2mL). (2S,4S)-4-amino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethylpentanoicacid ethyl ester (HCl salt; 120 mg, 0.3 mmol) was combined with1-butanol (3 mL) and 4 M of HCl in 1,4-dioxane (3 mL) and stirred at 60°C. for 2 hours. The solvent were evaporated and azeotroped with toluene(2×). and the product was dissolved in Et₃N (155 μL) and DCM, thencombined with the chloro-oxo-acetic acid t-butyl ester. The resultingmixture was stirred for 20 minutes at room temperature. The solvent wasevaporated and the residue was redissolved in 1:1 TFA:DCM, and stirredfor 20 minutes at 40° C. AcOH was added and the product was purified bypreparative HPLC to yield the title compound (30 mg, purity 95%). MS m/z[M+H]⁺ calc'd for C₂₄H₂₈FNO₆, 446.19; found 446.4.

B.(2S,4S)-5-(2′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-4-(oxalylamino)pentanoicAcid

Ethyl oxalyl chloride (13.8 μL, 0.1 mmol) and DIPEA (39.2 μL, 0.2 mmol)were combined with(2S,4S)-4-amino-5-(2′-fluorobiphenyl-4-yl)-2-hydroxymethylpentanoic acidethyl ester (HCl salt; 43 mg, 0.1 mmol) dissolved in DCM (0.9 mL). Themixture was stirred at room temperature for 10 minutes, thenconcentrated under vacuum. 1 M aqueous LiOH (0.9 mL) and EtOH (0.9 mL)was added and the resulting mixture was stirred at room temperature for1 hour. The reaction mixture was quenched with AcOH and the solvent wasevaporated. The residue was dissolved in AcOH/MeCN and purified bypreparative HPLC. The clean fractions were combined and lyophilized toyield the title compound (32.7 mg, purity 95%). MS m/z [M+H]⁺ calc'd forC₂₀H₂₀FNO₆, 390.13; found 390.2.

Example 8

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, thefollowing compound were prepared:

MS m/z: [M + H]⁺ Ex. R¹ R⁶ Formula calcd found 1 —CH₂CH₃ F C₂₂H₂₄FNO₆418.16 418.4 2 H F C₂₀H₂₀FNO₆ 390.13 390.4 3 H Cl C₂₀H₂₀ClNO₆ 406.10406.4

1.(2S,4S)-5-(3′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-4-(oxalylamino)pentanoicacid ethyl ester

-   2.    (2S,4S)-5-(3′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-4-(oxalylamino)pentanoic    acid-   3.    (2S,4S)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxymethyl-4-(oxalylamino)pentanoic    acid

MS m/z: [M + H]⁺ Ex. R¹ R⁶ Formula calcd found 4 H F C₂₀H₂₀FNO₆ 390.13390.2

-   4.    (2S,4S)-5-(4′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-4-(oxalylamino)pentanoic    acid

MS m/z: [M + H]⁺ Ex. R¹ R⁵ Formula calcd found 5 H Cl C₂₀H₂₀ClNO₆ 406.10406.0 6 H Cl C₂₀H₂₀ClNO₆ 406.10 406.0

-   5.    (2S,4S)-5-(3-Chlorobiphenyl-4-yl)-2-hydroxymethyl-4-(oxalylamino)pentanoic    acid-   6.    (2R,4S)-5-(3-Chlorobiphenyl-4-yl)-2-hydroxymethyl-4-(oxalylamino)pentanoic    acid

Example 9 A.(2S,4R)-5-Biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-(oxalylamino)pentanoicAcid

Ethyl oxalyl chloride (13.1 μL, 0.1 mmol) was combined with(2S,4R)-4-amino-5-biphenyl-4-yl-2-hydroxymethyl-2-methylpentanoic acidethyl ester (40 mg, 0.1 mmol) dissolved in DCM (0.3 mL) and a smallamount of DMF. The mixture was stirred at room temperature for 20minutes, then concentrated under vacuum. 1 M aqueous NaOH (117 μL) andTHF (1.5 mL) was added and the resulting mixture was stirred at roomtemperature for 30 minutes. The residue was dissolved in AcOH andpurified by preparative HPLC to yield the title compound (8 mg, purity95%). MS m/z [M+H]⁺ calc'd for C₂₁H₂₃NO₆, 386.15; found 386.0.

B.(2S,4R)-5-Biphenyl-4-yl-2-hydroxymethyl-2-methyl-4-(oxalylamino)pentanoicAcid Ethyl Ester

Oxalyl chloride (12.4 μL. 0.1 mmol) and t-butyl alcohol (13.1 μL) werecombined in ether (0.3 mL) under nitrogen at 0° C. The resulting mixturewas stirred for 30 minutes at room temperature. The solvent wasevaporated under vacuum to form chloro-oxo-acetic acid t-butyl ester,which was then dissolved in DCM (0.7 mL) and combined with(2S,4R)-4-amino-5-biphenyl-4-yl-2-hydroxymethyl-2-methylpentanoic acidethyl ester (33.4 mg, 98 μmol). Et₃N (43.6 μL,) at 0° C. was added andthe resulting mixture was stirred for 30 minutes at room temperature.The solvent were evaporated and the residue was dissolved in 1:1 TFA:DCMand stirred for 1 hour. AcOH was added and the product was purified bypreparative HPLC to yield the title compound (7 mg, purity 95%). MS m/z[M+H]⁺ calc'd for C₂₃H₂₇NO₆, 414.18; found 414.4.

Example 10(2S,4R)-5-(3′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-(oxalylamino)pentanoicAcid

Ethyl oxalyl chloride (9.1 μL, 0.1 mmol) was combined with(2S,4R)-4-amino-5-(3′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methylpentanoicacid (27 mg, 0.1 mmol) dissolved in DCM (0.2 mL) and a small amount ofDMF. The mixture was stirred at room temperature for 20 minutes. Thesolvent was evaporated and 10 M aqueous NaOH (81.5 μL), and THF (1.0 mL)was added and the resulting mixture was stirred at room temperature for30 minutes. The residue was dissolved in AcOH and purified bypreparative HPLC to yield the title compound (6 mg, purity 95%). MS m/z[M+H]⁺ calc'd for C₂₁H₂₂FNO₆, 404.14; found 404.4.

Example 11

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, thefollowing compounds were prepared:

MS m/z: [M + H]⁺ Ex. b R⁶ Formula calcd found 1 1 2′-F C₂₁H₂₂FNO₆ 404.14404.4 2 2 2′-F, 5′-Cl C₂₁H₂₁ClFNO₆ 438.10 438.2

-   1.    (2S,4R)-5-(2′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-(oxalylamino)pentanoic    acid-   2.    (2R,4R)-5-(5′-Chloro-2′-fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-(oxalyl-amino)pentanoic    acid

MS m/z: [M + H]⁺ Ex. R⁶ Formula calcd found 3 F C₂₁H₂₂FNO₆ 404.14 404.4

-   3.    (2S,4R)-5-(4′-Fluorobiphenyl-4-yl)-2-hydroxymethyl-2-methyl-4-(oxalylamino)pentanoic    acid

MS m/z: [M + H]⁺ Ex. R⁶ Formula calcd found 4 Cl C₂₅H₃₀ClNO₆ 476.18476.2

-   4.    (2S,4R)-5-(3′-Chlorobiphenyl-4-yl)-2-hydroxymethyl-4-(isobutoxyoxalyl-amino)-2-methylpentanoic    acid

Example 123-(N-Biphenyl-4-ylmethyl-N′-oxalylhydrazino)-2-hydroxy-2-methylpropionicAcid

3-(N-Biphenyl-4-ylmethyl-N′-t-butoxycarbonylhydrazino)-2-hydroxy-2-methylpropionicacid methyl ester (0.1 g, 241 μmol) was dissolved in DCM (1.0 mL), thenTFA (1.0 mL) was added and the mixture was stirred at room temperaturefor 1 hour. The mixture was concentrated and the residue was dissolvedin DMF (2.00 mL). DIPEA (126 μL, 724 μmol) was added followed by ethyloxalyl chloride (29.6 μL, 265 μmol) and the resulting mixture wasstirred at room temperature until the reaction was complete (˜3 hours).The mixture was concentrated and the residue was dissolved in THF (1.5mL), then lithium hydroxide monohydrate (101 mg, 2.4 mmol) in water(1.50 mL) was added and the mixture was stirred at room temperature for30 minutes. The reaction was quenched with AcOH and the solution wasconcentrated. The crude product was purified by preparative HPLC (10-70%MeCN/H₂O) to yield the title compound (10.9 mg, purity 95%). MS m/z[M+H]⁺ calc'd for C₁₉H₂₀N₂O₆, 373.13; found 373.2.

Example 13(R)-3-[N-(3′-Chlorobiphenyl-4-ylmethyl)-N′-oxalylhydrazino]-2-hydroxypropionicAcid

(R)-3-[N-(3′-Chlorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxy-propionicacid ethyl ester (70 mg, 0.2 mmol) was dissolved in DCM (1.5 mL),followed by the addition of ethyl oxalyl chloride (24.7 μL, 221 μmol)and DIPEA (69.9 μL, 401 μmol). The mixture was stirred at roomtemperature until the reaction was complete (˜10 minutes). The mixturewas the concentrated under vacuum. 1 M aqueous lithium hydroxide (1.6mL, 1.6 mmol) and EtOH (1.5 mL) was added and the mixture was stirred atroom temperature until the reaction was complete (˜2 hours). Thereaction was quenched with AcOH and the solvent was evaporated. Theresidue was dissolved in AcOH/MeCN and purified by preparative HPLC. Theclean fractions were combined and lyophilized to yield the titlecompound (8.3 mg, purity 95%). MS m/z [M+H]⁺ calc'd for C₁₈H₁₇ClN₂O₆,393.08; found 393.2.

Example 14 A.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-oxalylhydrazino]-2-hydroxypropionicAcid

Oxalic acid(R)-2-[N-(4-bromobenzyl)-N′-ethoxyoxalylhydrazino]-1-ethoxycarbonylethylester ethyl ester (675 mg, 1.3 mmol) was combined with5-chloro-2-fluorophenylboronic acid (273 mg, 1.6 mmol) and K₂CO₃ (541mg, 3.9 mmol) in EtOH (4.6 mL, 78.3 mmol) and water (1.2 mL, 65.2 mmol).The resulting mixture was placed under nitrogen atmosphere andSilicaCat®DPP-Pd (0.28 mmol/g loading; 466 mg, 130 nmol) was then added.The mixture was microwaved at 120° C. for 10 minutes, then filtered andevaporated under reduced pressure. The residue was purified bypreparative HPLC to yield the title compound (40 mg). MS m/z [M+H]⁺calc'd for C₁₈H₁₆ClFN₂O₆, 411.07; found 411.0.

B.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-oxalylhydrazino]-2-hydroxypropionicAcid Ethyl Ester

A ˜1M solution of t-butyl oxalyl chloride in DCM (136 μL) was added to astirred solution of(R)-3-[N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid ethyl ester (HCl salt; 55.0 mg, 136 μmol) in DCM (1.3 mL, 20 mmol)at 0° C. After stirring at room temperature for 2 hours, DIPEA (11.9 μL,68 μmol) in a DCM solution (80 μL) was added dropwise. After one minute,additional DIPEA (10 μL) in DCM (80 μL) was added, and the mixture wasstirred at room temperature overnight. The mixture was concentrated andthe resulting residue was purified by purified by flash chromatography(4 g silica gel, 0-100% EtOAc/hexanes). The desired fractions werecombined and concentrated to yield a colorless oil (60 mg). A portion ofthis oil (20 mg) was treated with a 1:1 mixture of DCM:TFA (0.2 mL) atroom temperature for 20 minutes. The mixture was concentrated, theresidue was dissolved in 50% water/AcOH (1.5 mL), filtered, and purifiedby reverse phase preparative to yield the title compound (10 mg) as aTFA salt. MS m/z [M+H]⁺ calc'd for C₂₀H₂₀ClFN₂O₆, 439.10; found 439.4.

C.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-isobutoxyoxalylhydrazino]-2-hydroxypropionicAcid

Chloro-oxo-acetic acid isobutyl ester was prepared by adding oxalylchloride (21 μL, 252 μmol) to a solution of isobutanol (21 μL, 226 μmol)in ether (206 μL, 2.0 mmol). The mixture was stirred at room temperaturefor 15 min and then evaporated.

The chloro-oxo-acetic acid isobutyl ester was then added to a solutionof(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid 2-oxo-2-phenylethyl ester (23.0 mg, 50 μmol) in DCM (413 μL, 6.4mmol) at 0° C. The resulting mixture was stirred at 0° C. for 15minutes. Saturated aqueous NaHCO₃ was then added and the layers wereseparated. The aqueous layer was extracted with DCM. The DCM layers werecombined, dried over MgSO₄ and concentrated to yield a clear yellowliquid. Zinc (164 mg, 2.5 mmol) was added to a solution of this yellowliquid in AcOH (172 μL, 3.0 mmol) and the mixture was stirred at roomtemperature for 10 minutes. The mixture was filtrated using AcOH andwater, the solvents were evaporated in vacuo, and the residue waspurified by preparative HPLC to yield the title compound (9.0 mg). MSm/z [M+H]⁺ calc'd for C₂₂H₂₄ClFN₂O₆, 467.13; found 467.1.

D.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(2,2-difluoropropoxyoxalyl)-hydrazino]-2-hydroxypropionicAcid

2,2-Difluoropropyl oxalyl chloride was prepared by adding oxalylchloride (21 μL, 252 μmol) to a solution of 2,2-difluoropropanol (21.8mg, 226 μmol) in ether (206 μL, 2.0 mmol). The mixture was stirred atroom temperature for 15 minutes and then evaporated.

The 2,2-difluoropropyl oxalyl chloride was then added to a solution of(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid 2-oxo-2-phenylethyl ester (23.0 mg, 50 μmol) in DCM (413 μL, 6.4mmol) at 0° C. The resulting mixture was stirred at 0° C. for 15minutes. Saturated aqueous NaHCO₃ was then added and the layers wereseparated. The aqueous layer was extracted with DCM. The DCM layers werecombined, dried over MgSO₄, and concentrated to yield a clear yellowliquid. Zinc (164 mg, 2.5 mmol) was added to a solution of this yellowliquid in AcOH (172 μL, 3.0 mmol) and the mixture was stirred at roomtemperature for 10 minutes. The mixture was filtrated using AcOH andwater, the solvents were evaporated in vacuo, and the residue waspurified by preparative HPLC to yield the title compound (1.1 mg). MSm/z [M+H]⁺ calc'd for C₂₁H₂₀ClF₃N₂O₆, 489.10; found 489.0.

E.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-oxalyl-hydrazino]-2-hydroxypropionicacid 5-Methyl-2-oxo-[1,3]dioxol-4-ylmethyl Ester

To a solution of(R)-3-[N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid 5-methyl-2-oxo-[1,3]dioxol-4-ylmethyl ester (350 mg, 780 μmol) inanhydrous DCM (15 mL) was added t-butyl oxalyl chloride (193 mg, 1.2mmol) and DIPEA (302 mg, 2.3 mmol) at 0° C. The resulting mixture wasstirred at room temperature for 5 hours. The mixture was then washedwith saturated aqueous NaCl (2×30 mL) and extracted with DCM (3×50 mL).The combined organic layers were dried over anhydrous Na₂SO₄, andconcentrated in vacuo to yield a white solid (300 mg). LC-MS: 523[M-tBu+H]⁺.

This solid (100 mg, 170 μmol) was dissolved in TFA (5 mL) and DCM (15mL). The resulting mixture was stirred overnight. The mixture wasevaporated in vacuo, and the residue was purified by preparative HPLC toyield the g title compound as a white solid (20 mg. LC-MS: 523.1 [M+H]⁺.¹H-NMR: (DMSO-d₆): δ 2.14 (s, 3H), 3.17-3.16 (m, 2 H), 4.11-4.08 (m, 2H), 4.26 (br, 1 H), 4.98 (br, 2 H), 5.50 (br, 1 H), 7.58-7.36 (m, 7 H),9.94 (s, 1 H), 13.8 (br, 1 H).

F.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-ethoxyoxalylhydrazino]-2-hydroxypropionicAcid

Ethyl oxalyl chloride (12.4 μL, 111 μmol) was added to a solution of(R)-3-[N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid 2-oxo-2-phenylethyl ester (23.0 mg, 50 μmol) in DCM (413 μL, 6.4mmol) at 0° C. and the resulting mixture was stirred at 0° C. for 15minutes. Saturated aqueous NaHCO₃ (1 mL) was then added and the layerswere separated. The aqueous layer was extracted with DCM (2×2 mL). TheDCM layers were combined, dried over MgSO₄, and concentrated. Zinc (164mg, 2.5 mmol) was added to a solution of this residue in AcOH (172 μL,3.0 mmol) and the resulting mixture was stirred at room temperature for10 minutes. The mixture was filtrated and the residue was purified bypreparative HPLC to yield the title compound (10 mg). MS m/z [M+H]⁺calc'd for C₂₀H₂₀ClFN₂O₆, 439.10; found 439.1.

G.(R)-3-[N-(5′-Chloro-2′-fluoro-iphenyl-4-ylmethyl)-N′-oxalylhydrazino]-2-hydroxypropionicAcid 2,2-Difluoropropyl Ester

(R)-3-[N′-t-Butoxyoxalyl-N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]2-hydroxy-propionicacid (15.0 mg, 32 μmol) was combined with HOBt (26.0 mg, 193 μmol) andEDC (34 μL, 0.2 mmol) in DCM (0.2 mL, 4 mmol). The solution was stirredfor 10 minutes and 2,2-difluoropropanol (24.7 mg, 257 μmol) was added.The reaction was stirred at room temperature and monitored forcompletion. After 2 hours, the mixture was concentrated by rotaryevaporation and the solvent was removed in vacuo. The resulting residuewas dissolved in DCM (124 μL, 1.9 mmol). TFA (124 μL, 1.6 mmol) wasadded, and the resulting mixture was stirred for 2 hours. The solventwas removed in vacuo and the residue was purified by preparative HPLC toyield the title compound (2.2 mg). MS m/z [M+H]⁺ calc'd forC₂₁H₂₀ClF₃N₂O₆, 489.10; found 489.1.

H.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-oxalylhydrazino]-2-hydroxypropionicAcid Isobutyl Ester

To a mixture of(R)-3-[N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid ethyl ester (HCl salt; 500.0 mg, 1.3 mmol) in DCM (6.0 mL, 94 mmol)at room temperature was added di-t-butyldicarbonate (342 μL, 1.5 mmol)and DIPEA (216 μL, 1.3 mmol). After stirring at room temperatureovernight, the mixture was concentrated and the residue was purified byflash chromatography (12 g silica gel, 0-50% EtOAc/hexanes). The desiredfractions were combined and concentrated to give a light yellowish oil.This oily residue was dissolved in MeOH (6.0 mL, 150 mmol) and water(1.0 mL, 56 mmol), then treated with LiOH monohydrate (104 mg, 2.5 mmol)at room temperature for 30 minutes. The mixture was concentrated and theresidue was diluted with water (2.0 mL) and EtOAc (10.0 mL), thenacidified with 1N aqueous HCl until pH˜2.0 with vigorous stirring. Theorganic layer was washed with saturated aqueous NaCl (2×2.0 mL), driedover Na₂SO₄, filtered, and concentrated to give Compound 1 as a whitesolid (528.6 mg).

Compound 1 (65.0 mg, 148 μmol) was dissolved in isobutyl alcohol (684μL, 7.4 mmol). A solution of 4.0 M HCl in 1,4-dioxane (1.2 mL, 4.9 mmol)was added and the resulting mixture was stirred at room temperature for2 hours, then at 60° C. for an other couple of hours, until the reactionwas complete. The solvent was removed in vacuo to yield Compound 2,which was used without further purification.

t-Butyl oxalyl chloride was prepared by adding oxalyl chloride (63 nL,741 nmol) to a solution of t-butyl alcohol (43 nL, 444 nmol) in ether(778 nL, 7.4 mmol). The mixture was stirred at room temperature for 15minutes, then concentrated in vacuo. Compound 2 (58.5 mg, 148 nmol) wasdissolved in DCM (570 nL, 8.9 mmol) and t-butyl oxalyl chloride wasadded. The resulting mixture was stirred at room temperature for 30minutes and then concentrated in vacuo. The residue was dissolved in a1:1 DCM:TFA solution and stirred at room temperature for 1 hour. Thesolvent was removed in vacuo and the residue was purified by preparativeHPLC to yield the title compound (8.5 mg). MS m/z [M+H]⁺ calc'd forC₂₂H₂₄ClFN₂O₆, 467.13; found 467.0.

I.(R)-3-[N′-t-Butoxyoxalyl-N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicAcid Ethyl Ester

To a solution of(R)-3-[N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid ethyl ester (200 mg, 0.5 mmol) in DCM (2.0 mL) was added dropwise asolution of t-butyl oxalyl chloride (165 mg, 1.0 mmol) at 0° C. undernitrogen. The resulting mixture was stirred for 5 minutes and then DIPEA(130 mg, 1.0 mmol). was added dropwise. The solvent was removed byevaporation, and the residue was purified by column chromatography(petroleum ether/EtOAc=4:1) to yield the title compound as a yellowliquid (144 mg). LC-MS: 495 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): δ 1.30 (t,J=7.1 Hz, 3H), 1.56 (s, 9H), 3.37-3.24 (m, 2H), 4.27-4.16 (m, 4H),4.38-4.30 (m, 1H), 7.14-7.09 (m, 1H), 7.30-7.28 (m, 1H), 7.48-7.41 (m,3H), 7.56-7.50 (m, 2H), 8.05 (s, 1H).

J.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-oxalythydrazino]-2-hydroxy-propionicAcid Ethoxycarbonyloxymethyl Ester

A mixture of(R)-3-[N′-t-butoxyoxalyl-N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid (270 mg, 580 μmol), chloromethyl ethyl carbonate (160 mg, 1.16mmol), NaI (174 mg, 1.2 mmol) and 2,6-dimethylpyridine (620 mg, 5.8mmol) in DMF (10 mL) was stirred at room temperature overnight. Themixture was poured into water (30 mL) and the mixture was then extractedwith EtOAc (3×30 mL). The combined organic layers were washed withsaturated aqueous NaCl (2×30 mL), dried over anhydrous Na₂SO₄, andconcentrated in vacuo. The crude Compound 1 (300 mg) was used withoutpurification. LC-MS: 569 [M+H]⁺.

TFA (1.0 mL) was added dropwise at room temperature to a solution ofCompound 1 (300 mg, 530 μmol) in DCM (5 mL). The resulting mixture wasstirred for 2 hours at room temperature, and the solvent was thenremoved. The residue was purified by column chromatography (DCM/MeOH,10:1) to yield the title compound as a yellow liquid (10 mg). LC-MS:512.9 [M+H]⁺. ¹H NMR (400 MHz, MeOD) δ 1.28 (t, J=7.3 Hz, 3H), 3.24-3.28(m, 2H), 4.18-4.20 (m, 4H), 4.41 (br, 1H), 5.80 (dd, J=11.6, 5.8 Hz,2H), 7.22 (d, J=10.1 Hz, 1H), 7.34-7.40 (m, 1H), 7.48-7.51 (m, 5H).

K. Butyric Acid(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-oxalylhydrazino]-2-hydroxypropionyloxymethylEster

A mixture of (R)-3-[N′-t-butoxyoxalyl-N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid (300 mg, 430 mmol), chloromethyl butyrate (175 mg, 1.3 mmol), NaI(192 mg, 1.3 mmol) and 2,6-dimethylpyridine (680 mg, 6.4 mmol) in DMF(10 mL) was stirred at room temperature overnight. The mixture waspoured into water (30 mL) and the mixture was then extracted with EtOAc(3×20 mL). The organic layer was separated, washed with saturatedaqueous NaCl (30 mL), dried over anhydrous Na₂SO₄, and concentrated invacuo. The crude Compound 1 (300 mg) was used without purification.LC-MS: 567[M+H]⁺.

TFA (1.0 mL) was added dropwise at room temperature to a solution ofCompound 1 (300 mg, 464 μmol) in DCM (5 mL). The resulting mixture wasstirred for 2 hours at room temperature, and the solvent was thenremoved. The residue was purified by column chromatography (DCM/MeOH,10:1) to yield the title compound as a yellow oil (21 mg). LC-MS:511.1[M+H]⁺. ¹H NMR (400 MHz, MeOD) δ 0.94 (t, J=7.4 Hz, 3H), 1.62 (dd,J=14.8, 7.4 Hz, 2H), 2.33 (t, J=7.3 Hz, 2H), 3.28 (d, J=6.1 Hz, 2H),4.14 (q, J=13.2 Hz, 2H), 4.38 (dd, J=6.0, 4.2 Hz, 1H), 5.80 (br, 2H),7.16-7.26 (m, 1H), 7.33-7.40 (m, 1H), 7.47-7.52 (m, 5H).

L.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-oxalylhydrazino]-2-hydroxypropionicAcid Acetoxymethyl Ester

A mixture of(R)-3-[N′-t-butoxyoxalyl-N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)hydrazino]-2-hydroxypropionicacid (300 mg, 640 mmol), bromomethyl acetate (196 mg, 1.3 mmol), NaI(192 mg, 1.3 mmol) and 2,6-dimethylpyridine (680 mg, 6.4 mmol) in DMF(10 mL) was stirred at room temperature overnight. The mixture waspoured into water (30 mL) and the mixture was then extracted with EtOAc(3×20 mL). The organic layer was separated, washed with saturatedaqueous NaCl (30 mL), dried over anhydrous Na₂SO₄, and concentrated invacuo. The crude Compound 1 (300 mg) was used without purification.LC-MS: 539 [M+H]⁺.

TFA (1.0 mL) was added dropwise at room temperature to a solution ofCompound 1 (300 mg, 550 μmol) in DCM (5 mL). The resulting mixture wasstirred for 2 hours at room temperature, and the solvent was thenremoved. The residue was purified by column chromatography (DCM/MeOH,10:1) to yield the title compound as a yellow oil (15 mg). LC-MS: 482.9[M+H]⁺. ¹H NMR (400 MHz, MeOD) δ 2.07 (s, 3H), 3.25-3.28 (m, 2H), 4.14(q, J=13.2 Hz, 2H), 4.38 (t, J=5.9 Hz, 1H), 5.88-5.71 (m, 2H), 7.25-7.17(m, 1H), 7.41-7.31 (m, 1H), 7.70-7.46 (m, 5H).

Example 15(2R,4S)-5-Biphenyl-4-yl-2-hydroxy-5-methyl-4-(oxalylamino)hexanoic Acid

(2R,4S)-4-Amino-5-biphenyl-4-yl-2-hydroxy-5-methylhexanoic acid ethylester (70 mg, 0.2 mmol) was dissolved in DCM (5 mL) and stirred for 2minutes, followed by the addition of ethyl oxalyl chloride (23 μL, 0.2mmol) and DIPEA (79 mg, 0.6 mmol). The mixture was stirred at roomtemperature for 1 hour, then evaporated under reduced pressure. Themixture was the concentrated under vacuum. The residue was dissolved inEtOH, and sufficient equivalents of 10N NaOH were added to make thesolution basic. The reaction was monitored over 1 hour until finaldeprotection was complete. The solution was acidified with an equalvolume of AcOH and evaporated under reduced pressure. The product wasthen purified using reverse phase chromatography (gradient of 10-70%MeCN to yield the title compound (37 mg, purity 95%). MS m/z [M+H]⁺calc'd for C₂₁H₂₃NO₆, 386.15; found 386.4.

Example 16(2S,4S)-5-Biphenyl-4-yl-2-hydroxymethyl-5-methyl-4-(oxalylamino)hexanoicAcid

(2S,4S)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-5-methylhexanoic acidethyl ester (HCl salt; 40 mg, 0.1 mmol) was dissolved in DCM and DMF (1mL), followed by the addition of ethyl oxalyl chloride (17 μL, 0.2 mmol)and DIPEA (53.3 μL, 0.3 mmol). The mixture was stirred at roomtemperature until the reaction was complete (˜5 minutes). The reactionwas quenched with water. The product extracted with EtOAc and theresulting organic layer was concentrated. 1 M aqueous lithium hydroxide(1.0 mL, 1.0 mmol) and EtOH (2.0 mL) was added and the mixture wasstirred at room temperature until the reaction was complete (˜2 hours).The reaction was quenched with AcOH and the product was purified bypreparative HPLC. The clean fractions were combined and lyophilized toyield the title compound (19 mg, purity 95%). MS m/z [M+H]⁺ calc'd forC₂₂H₂₅NO₆, 400.17; found 400.2.

Additional compounds of the invention can be prepared using thefollowing starting materials:

(R)-4-Amino-5-biphenyl-4-yl-2-hydroxy-2-methyl-pentanoic Acid EthylEster

To a solution of (R)-3-biphenyl-4-yl-2-t-butoxycarbonylamino-propionicacid (50 g, 0.1 mol), Meldrum's acid (23.3 g, 0.2 mol) and DMAP (27.8 g,0.2 mol) in anhydrous DCM (500 mL) was added a solution of DCC (33.3 g,0.2 mol) in anhydrous DCM (200 mL) over 1 hour at −5° C. under nitrogen.The mixture was stirred at −5° C. for 8 hours, then refrigeratedovernight, during which tiny crystals of dicyclohexylurea precipitated.After filtration, the mixture was washed with 5% KHSO₄ (4×200 mL),saturated aqueous NaCl (200 mL) and dried under refrigeration with MgSO₄overnight. The resulting solution was evaporated to yield crude Compound1 as a light yellow solid (68 g). LC-MS: 490 [M+Na], 957 [2M+Na].

To a solution of crude Compound 1 (68 g, 0.1 mol) in anhydrous DCM (1 L)was added AcOH (96.8 g, 1.6 mol) at −5° C. under nitrogen. The mixturewas stirred at −5° C. for 0.5 hour, then NaBH₄ (13.9 g, 0.4 mol) wasadded in small portions over 1 hour. After stirring at −5° C. foranother 1 hour, saturated aqueous NaCl (300 mL) was added. The organiclayer was washed with saturated aqueous NaCl (2×300 mL) and water (2×300mL), dried over MgSO₄, filtered, and concentrated to give the crudeproduct which was further purified by chromatography (hexanes:EtOAc=5:1)to yield Compound 2 as a light yellow solid (46 g). LC-MS: 476 [M+Na],929 [2M+Na].

To a solution of Compound 2 (46 g, 0.1 mol) in tertiary butyl alcohol(100 mL) was added dimethylmethyleneimmonium iodide (46.3 g, 0.3 mol) atroom temperature under nitrogen. The mixture was heated to 65° C. andstirred at this temperature for 16 hours. After filtration, the filtratewas concentrated to give the crude product which was further purified bychromatography (hexanes:EtOAc=20:110:1) to yield Compound 3 as a lightyellow solid) (18 g). LC-MS: 460 [M+Na], 897 [2M+Na].

To a solution of Compound 3 (18 g, 44 mmol) in acetone (430 mL) andwater (22 mL) was added Sudan Red as indicator. Ozone atmosphere wasintroduced into the mixture at 0° C. until the red color of Sudan Reddisappeared. Dimethyl sulfide (45 mL) was added and the mixture wasstirred at room temperature overnight. The mixture was then concentratedand the residual was purified by chromatography (hexanes:EtOAc=15:1˜7:1)to yield Compound 4 as a light yellow solid (9.5 g). LC-MS: 434 [M+H],845 [2M+H].

To a solution of Compound 4 (9.5 g, 23 mmol) in anhydrous THF (120 mL)was added a solution of methylmagnesium bromide in THF (9.2 mL, 28 mmol)at −70° C. under nitrogen. The mixture was stirred at −60° C. for 3hours and the reaction was then quenched with saturated aqueous NH₄Cl(50 mL). The organic layer was separated and dried over

MgSO₄. The mixture was then concentrated and the residual was purifiedby chromatography (hexanes:EtOAc=10:1˜5:1) to yield Compound 5 as an oil(7.9 g). LC-MS: 450 [M+H], 877 [2M+H].

To a solution of Compound 5 (7.9 g, 18.4 mmol) in anhydrous DCM (300 mL)was pumped HCl atmosphere at 0° C. for 6 hours. The mixture was thenconcentrated and the residue was washed with anhydrous Et₂O to yield thetitle compound as a white solid HCl salt (5.8 g). LC-MS: 364 [M+H], 727[2M+H]. ¹H NMR (300 MHz, DMSO): δ8.00-7.97 (d, 4H), 7.67-7.62 (m, 6H),7.47-7.28 (m, 8H), 6.32 (s, 1H), 6.09 (s, 1H), 4.13-4.06 (m, 2H),3.95-3.78 (m, 2H), 3.60 (s, 1H), 3.22-3.08 (m, 3H), 2.95-2.65 (m, 2H),1.99-1.79 (m, 4H), 1.30-0.87 (m, 9H).

(R)-4-Amino-5-biphenyl-4-yl-2,2-dimethyl-pentanoic Acid Ethyl Ester

A solution of[(S)-1-biphenyl-4-ylmethyl-2-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-ethyl]-carbamicacid t-butyl ester (46 g, 0.1 mol) in anhydrous toluene (300 mL) wasrefluxed for 3 hours under nitrogen. After evaporation of the solvent,the residue was purified by chromatography (hexanes:EtOAc=10:1) to yieldCompound 1 as a light yellow solid (27 g). LC-MS: 374 [M+Na], 725[2M+Na].

To a solution of Compound 1 (6.2 g, 17.6 mmol) in anhydrous THF (100 mL)was added a solution of LiHMDS in THF (39 mL, 39 mmol) at −78° C. undernitrogen. The mixture was stirred at −78° C. for 2 hours, and thenmethyl iodide (7.5 g, 53 mmol) was added. After stirring for 0.5 hour at−78° C., the mixture was warmed to room temperature and stirred at roomtemperature for 3 hours. After the mixture cooled to −10° C., thereaction was quenched with saturated aqueous NH₄Cl (100 mL) andextracted with EtOAc (100 mL×4). The combined organic layers were washedwith saturated aqueous NaCl (300 mL), dried over MgSO₄, filtered, andconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=10:1) to yield Compound 2 as a lightyellow solid (5.7 g). LC-MS: 402 [M+Na], 781 [2M+Na].

To a solution of Compound 2 (5.7 g, 15 mmol) in acetone (120 mL) wasadded 1 M NaOH (60 mL, 60 mmol) at −5° C. under nitrogen. The mixturewas warmed to room temperature and stirred at room temperature for 20hours. The mixture was concentrated and the residual was diluted withwater (250 mL) and washed with EtOAc (150 mL). The pH of the aqueouslayer was adjusted to 2 with 6 M HCl at 0° C., and the solid wasfiltrated and dried in vacuo to yield the crude Compound 3 as a whitesolid (5 g). LC-MS: 420 [M+Na], 817 [2M+Na].

To a solution of crude Compound 3 (5 g, 12.7 mmol) in anhydrous EtOH(300 mL) was added SOCl₂ (13.4 mL, 190 mmol) at −30° C. under nitrogen.The mixture was warmed to room temperature and stirred for 20 hours atroom temperature. The mixture was concentrated, and the residual waswashed with anhydrous Et₂O to yield the title compound as a white solidHCl salt (3.7 g). LC-MS: 326 [M+H], 651 [2M+H]. ¹H NMR (300 MHz, DMSO):δ7.86 (s, 3H), 7.67-7.64 (m, 4H), 7.49-7.33 (m, 5H), 4.09-3.97 (m, 2H),3.42 (m, 1H), 2.90-2.80 (m, 2H), 1.88-1.84 (m, 2H), 1.17-1.12 (m, 9H).

1-((R)-2-Amino-3-biphenyl-4-yl-propyl)-cyclopropanecarboxylic Acid

Into a flask containing BOC-D-4,4′-biphenylalanine (11.3 g, 33.1 mmol,1.0 eq.), 4-dimethylaminopyridine (6.5 g, 53.0 mmol, 1.6 eq.),2,2-dimethyl-1,3-dioxane-4,6-dione (5.3 g, 36.4 mmol, 1.1 eq.) in DCM(100 mL) was added 1 M of DCC in DCM (38.1 mL) at 0° C. over 30 minutes.The mixture was maintained at 0° C. for 6 hours and the resultingprecipitate was filtered off. The filtrate was washed with aqueous 10%KHSO₄ (2×50 mL) then dried. The solution was acidified with AcOH (20 mL)at 0° C. and sodium borohydride (3.1 g, 82.7 mmol, 2.5 eq.) was addedover 30 minutes in 3 portions. The mixture was maintained at 0° C. for 3hours, washed with water and dried, then concentrated under vacuum. Thecrude material was purified by chromatography (0-40% EtOAc/hexanesgradient). Eschenmoser's salt (15.9 g, 86.0 mmol) in t-butyl alcohol (70mL) was added and the resulting mixture was stirred at 65° C. overnight.The mixture was concentrated and Et₂O (10 mL) was added. The organicsolution was then washed with saturated aqueous NaHCO₃ (10 mL) and 10%KHSO₄ (10 mL). The organic solution was dried over Na₂SO₄ andconcentrated. The crude product was purified by chromatography (0-40%EtOAc/hexanes gradient) to yield Compound 1 (3.3 g).

Trimethylsufoxonium iodide (2.0 g, 9.2 mmol, 1.0 eq.) in dimethylsulfoxide (50 mL) was combined with NaH (366 mg, 9.2 mmol, 1.1 eq.) amdstirred for 15 minutes at room temperature. To this was added Compound 1(3.6 g, 8.3 mmol, 1.0 eq) dissolved dimethyl sulfoxide (50 mL). Theresulting mixture was stirred at room temperature overnight. Thesolution was mixed with saturated aqueous NaCl (50 mL) and extractedwith EtOAc (3×10 mL), and the organic layer was washed with saturatedaqueous NaCl (2×50 mL) and dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the crude reaction was purified bychromatography (0-40% EtOAc/hexanes gradient) to yield Compound 2,1-((R)-3-biphenyl-4-yl-2-t-butoxycarbonylaminopropyl)-cyclopropanecarboxylicacid t-butyl ester. TFA (200 μL) and DCM (500 μL) were added and theresulting mixture was stirred for 30 minutes. The solvent was evaporatedunder vacuum and azeotroped with toluene (2×) to obtain the titlecompound.

Assay 1 In Vitro Assays for the Quantitation of Inhibitor Potencies atHuman and Rat NEP, and Human ACE

The inhibitory activities of compounds at human and rat neprilysin (EC3.4.24.11; NEP) and human angiotensin converting enzyme (ACE) weredetermined using in vitro assays as described below.

Extraction of NEP Activity from Rat Kidneys

Rat NEP was prepared from the kidneys of adult Sprague Dawley rats.Whole kidneys were washed in cold phosphate buffered saline (PBS) andbrought up in ice-cold lysis buffer (1% Triton X-114, 150 mM NaCl, 50 mMtris(hydroxymethyl) aminomethane (Tris) pH 7.5; Bordier (1981) J. Biol.Chem. 256: 1604-1607) in a ratio of 5 mL of buffer for every gram ofkidney. Samples were homogenized on ice using a polytron hand heldtissue grinder. Homogenates were centrifuged at 1000×g in a swingingbucket rotor for 5 minutes at 3° C. The pellet was resuspended in 20 mLof ice cold lysis buffer and incubated on ice for 30 minutes. Samples(15-20 mL) were then layered onto 25 mL of ice-cold cushion buffer (6%w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, Triton X-114),heated to 37° C. for 3-5 minutes and centrifuged at 1000×g in a swingingbucket rotor at room temperature for 3 minutes. The two upper layerswere aspirated off, leaving a viscous oily precipitate containing theenriched membrane fraction. Glycerol was added to a concentration of 50%and samples were stored at −20° C. Protein concentrations werequantitated using a BCA detection system with bovine serum albumin (BSA)as a standard.

Enzyme Inhibition Assays

Recombinant human NEP and recombinant human ACE were obtainedcommercially (R&D Systems, Minneapolis, Minn., catalog numbers 1182-ZNand 929-ZN, respectively). The fluorogenic peptide substrateMca-D-Arg-Arg-Leu-Dap-(Dnp)-OH (Medeiros et al. (1997) Braz. J. Med.Biol. Res. 30:1157-62; Anaspec, San Jose, Calif.) andAbz-Phe-Arg-Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochemistry39:8519-8525; Bachem, Torrance, Calif.) were used in the NEP and ACEassays respectively.

The assays were performed in 384-well white opaque plates at 37° C.using the fluorogenic peptide substrates at a concentration of 10 μM inAssay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethyleneglycol sorbitan monolaurate (Tween-20), 10 μM ZnSO₄; ACE: 50 mM HEPES,pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO₄). The respective enzymeswere used at concentrations that resulted in quantitative proteolysis of1 μM of substrate after 20 minutes at 37° C.

Test compounds were assayed over the range of concentrations from 10 μMto 20 pM. Test compounds were added to the enzymes and incubated for 30minute at 37° C. prior to initiating the reaction by the addition ofsubstrate. Reactions were terminated after 20 minutes of incubation at37° C. by the addition of glacial acetic acid to a final concentrationof 3.6% (v/v).

Plates were read on a fluorometer with excitation and emissionwavelengths set to 320 nm and 405 nm, respectively Inhibition constantswere obtained by nonlinear regression of the data using the equation(GraphPad Software, Inc., San Diego, Calif.):v=v ₀/[1+(I/K′)]where v is the reaction rate, v₀ is the uninhibited reaction rate, I isthe inhibitor concentration and K′ is the apparent inhibition constant.

Compounds of the invention were tested in this assay and found to havepK_(i) values at human NEP as follows. In general, either the prodrugcompounds did not inhibit the enzyme in this in vitro assay, or theprodrugs were not tested (n.d.) since activity would not be expected.

Ex. pK_(i)  1 7.0-7.9  2A ≧9  2B n.d.  2C n.d.  2D n.d.  2E n.d.  2F ≧9 2G 8.0-8.9  2H ≧9  2I 8.0-8.9  2J 7.0-7.9  2K n.d.  2L n.d.  2M n.d. 2N ≧9  2O n.d.  2P n.d.  2Q n.d.  2R n.d.  2S n.d.  2T ≧9  2U ≧9  2V ≧9 2W  3A ≧9  3B n.d.  3C n.d.  3D ≧9  4 8.0-8.9  5-1 ≧9  5-2 n.d.  5-3n.d.  5-4 n.d.  5-5 ≧9  5-6 n.d.  5-7 n.d.  5-8 n.d.  5-9 ≧9  5-10 n.d. 5-11 n.d.  5-12 n.d.  5-13 ≧9  5-14 ≧9  5-15 ≧9  5-16 n.d.  5-17 ≧9  6A≧9  6B n.d.  7A n.d.  7B ≧9  8-1 n.d.  8-2 ≧9  8-3 ≧9  8-4 8.0-8.9  8-58.0-8.9  8-6 7.0-7.9  9A 8.0-8.9  9B n.d. 10 8.0-8.9 11-1 8.0-8.9 11-2≧9 11-3 7.0-7.9 11-4 8.0-8.9 12 7.0-7.9 13 8.0-8.9 14A ≧9 14B n.d. 14C8.0-8.9 14D n.d. 14E n.d. 14F 8.0-8.9 14G n.d. 14H n.d. 14I n.d. 14Jn.d. 14K n.d. 14L n.d. 15 7.0-7.9 16 8.0-8.9 n.d. = not determined

Assay 2 Pharmacodynamic (PD) Assay for ACE and NEP Activity inAnesthetized Rats

Male, Sprague Dawley, normotensive rats are anesthetized with 120 mg/kg(i.p.) of inactin. Once anesthetized, the jugular vein, carotid artery(PE 50 tubing) and bladder (flared PE 50 tubing) catheters arecannulated and a tracheotomy is performed (Teflon Needle, size 14 gauge)to faciliate spontaneous respiration. The animals are then allowed a 60minute stablization period and kept continuously infused with 5 mL/kg/hof saline (0.9%) throughout, to keep them hydrated and ensure urineproduction. Body temperature is maintained throughout the experiment byuse of a heating pad. At the end of the 60 minute stabilization period,the animals are dosed intravenously (i.v.) with two doses of AngI (1.0μg/kg, for ACE inhibitor activity) at 15 minutes apart. At 15 minutespost-second dose of AngI, the animals are treated with vehicle or testcompound. Five minutes later, the animals are additionally treated witha bolus i.v. injection of atrial natriuretic peptide (ANP; 30 μg/kg).Urine collection (into pre-weighted eppendorf tubes) is startedimmediately after the ANP treatment and continued for 60 minutes. At 30and 60 minutes into urine collection, the animals are re-challenged withAngI. Blood pressure measurements are done using the Notocord system(Kalamazoo, Mich.). Urine samples are frozen at −20° C. until used forthe cGMP assay. Urine cGMP concentrations are determined by EnzymeImmuno Assay using a commercial kit (Assay Designs, Ann Arbor, Mich.,Cat. No. 901-013). Urine volume is determined gravimetrically. UrinarycGMP output is calculated as the product of urine output and urine cGMPconcentration. ACE inhibition is assessed by quantifying the %inhibition of pressor response to AngI. NEP inhibition is assessed byquantifying the potentiation of ANP-induced elevation in urinary cGMPoutput.

Assay 3 In Vivo Evaluation of Antihypertensive Effects in the ConsciousSHR Model of Hypertension

Spontaneously hypertensive rats (SHR, 14-20 weeks of age) are allowed aminimum of 48 hours acclimation upon arrival at the testing site withfree access to food and water. For blood pressure recording, theseanimals are surgically implanted with small rodent radiotransmitters(telemetry unit; DSI Models TA11PA-C40 or C50-PXT, Data Science Inc.,USA). The tip of the catheter connected to the transmitter is insertedinto the descending aorta above the iliac bifurcation and secured inplace with tissue adhesive. The transmitter is kept intraperitoneallyand secured to the abdominal wall while closing of the abdominalincision with a non-absorbable suture. The outer skin is closed withsuture and staples. The animals are allowed to recover with appropriatepost operative care. On the day of the experiment, the animals in theircages are placed on top of the telemetry receiver units to acclimate tothe testing environment and baseline recording. After at least of 2hours baseline measurement is taken, the animals are then dosed withvehicle or test compound and followed out to 24 hours post-dose bloodpressure measurement. Data is recorded continuously for the duration ofthe study using Notocord software (Kalamazoo, Mich.) and stored aselectronic digital signals. Parameters measured are blood pressure(systolic, diastolic and mean arterial pressure) and heart rate.

Assay 4 In Vivo Evaluation of Antihypertensive Effects in the ConsciousDOCA-Salt Rat Model of Hypertension

CD rats (male, adult, 200-300 grams, Charles River Laboratory, USA) areallowed a minimum of 48 hours acclimation upon arrival at the testingsite before they are placed on a high salt diet. One week after thestart of the high salt diet (8% in food or 1% NaCl in drinking water), adeoxycorticosterone acetate (DOCA) pellet (100 mg, 90 days release time,Innovative Research of America, Sarasota, Fla.) is implantedsubcutaneously and unilateral nephrectomy is performed. At this time,the animals are also surgically implanted with small rodentradiotransmitters for blood pressure measurement (see Assay 3 fordetails). The animals are allowed to recover with appropriate postoperative care. Study design, data recording, and parameters measured issimilar to that described for Assay 3.

Assay 5 In Vivo Evaluation of Antihypertensive Effects in the ConsciousDahl/SS Rat Model of Hypertension

Male, Dahl salt sensitive rats (Dahl/SS, 6-7 weeks of age from CharlesRiver Laboratory, USA) are allowed at least 48 hours of acclimation uponarrival at the testing site before they were placed on a 8% NaCl highsalt diet (TD.92012, Harlan, USA) then surgically implanted with smallrodent radiotransmitters for blood pressure measurement (see Assay 3 fordetails). The animals are allowed to recover with appropriate postoperative care. At approximately 4 to 5 weeks from the start of highsalt diet, these animals are expected to become hypertensive. Once thehypertension level is confirmed, these animals are used for the studywhile continued with the high salt diet to maintain their hypertensionlevel. Study design, data recording, and parameters measured is similarto that described in Assay 3.

Comparative Example 1

(2R,4S)-5-Biphenyl-4-yl-2-methyl-4-(oxalyl-amino)-pentanoic Acid(Comparative Compound A; R=—C(O)—COOH)

(2R,4S)-4-Amino-5-biphenyl-4-yl-2-methyl-pentanoic acid ethyl ester (HClsalt; 527 mg, 0.2 mmol) and ethyl oxalyl chloride (18.4 μL, 1.1 eq) werecombined with DIPEA (52.2 μL, 0.3 mmol) in DMF (0.3 mL)/DCM (0.3 mL).The mixture was stirred at room temperature until the reaction wascomplete. The solvent was removed and the residue was dissolved in EtOH(750 μL) and 1 M aqueous NaOH (750 μL), and stirred at room temperatureovernight. The solvent was removed and the residue was purified bypreparative HPLC to yield Comparative Compound A (11.2 mg, 100% purity).MS m/z [M+H]⁺ calc'd for C₂₀H₂₁NO₅, 356.14; found 356.2.

(2R,4S)-5-Biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-pentanoicAcid (Comparative Compound B; R=—C(O)—(CH₂)₂—COOH)

(2R,4S)-5-Biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-pentanoicacid ethyl ester (Na salt; 400 mg, 923 μmol) was mixed with EtOH (7 mL,0.1 mol) then THF (6 mL, 0.1 mol). 1 M Aqueous NaOH (2.8 mL, 2.8 mmol)was then added and the resulting mixture was stirred at room temperaturefor 4 hours and was then concentrated. The product was purified bypreparative HPLC (10-60% MeCN:water w/0.5% TFA) to yield ComparativeCompound B (150 mg, 97% purity). MS m/z [M+H]⁺ calc'd for C₂₂H₂₅NO₅,384.17; found 384.6.

Comparative Compounds A and B were tested as described in Assay 1 andfound to have pK_(i) values at human NEP as follows:

Compound R pK_(i) Comparative Compound A —C(O)—COOH 8.2 ComparativeCompound B —C(O)—(CH₂)₂—COOH 8.2The data shows that Comparative Compounds A and B have the same pK_(i)values for the inhibition of NEP.

Comparative Example 2

(R)-5-Biphenyl-4-yl-4-(2-carboxy acetylamino)-2-hydroxypentanoic Acid(Comparative Compound C; R=—C(O)—CH₂—COOH)

(R)-4-Amino-5-biphenyl-4-yl-2-hydroxypentanoic acid ethyl ester (HClsalt; 60.3 mg, 0.2 mmol) and methyl malonyl chloride (21 μL, 0.2 mmol)were combined with DIPEA (84 μL, 0.5 mmol) in DMF (5 mL). The mixturewas stirred at room temperature until the reaction was complete (1hour). The solvent was removed and the residue was dissolved in MeOH (3mL) and 10N NaOH (250 μL), and stirred at 60° C. until the reaction wascomplete (1 hour). Glacial acetic acid (250 μL) was added and theproduct was evaporated under reduced pressure and purified bypreparative HPLC to yield Comparative Compound C (6.3 mg, 98% purity).MS m/z [M+H]⁺ calc'd for C₂₀H₂₁NO₆, 372.14; found 372.2.

(R)-5-Biphenyl-4-yl-4-(3-carboxypropionylamino)-2-hydroxypentanoic Acid(Comparative Compound D; R=—C(O)—(CH₂)₂—COOH)

(R)-4-Amino-5-biphenyl-4-yl-2-hydroxypentanoic acid ethyl ester (HClsalt; 60.3 mg, 0.2 mmol) and 3-(carbomethoxy)propionyl chloride (24 μL,0.2 mmol) were combined with DIPEA (84 μL, 0.5 mmol) in DMF (5 mL). Themixture was stirred at room temperature until the reaction was complete(1 hour). The solvent was removed and the residue was dissolved in MeOH(3 mL) and 10N NaOH (250 μL), and stirred at 60° C. until the reactionwas complete (1 hour). Glacial acetic acid (250 μL) was added and theproduct was evaporated under reduced pressure and purified bypreparative HPLC to yield Comparative Compound D (8.0 mg, 100% purity).MS m/z [M+H]⁺ calc'd for C₂H₂₃NO₆, 386.15; found 386.2.

(R)-5-Biphenyl-4-yl-4-(4-carboxybutyrylamino)-2-hydroxypentanoic Acid(Comparative Compound E; R=—C(O)—(CH₂)₃—COOH)

(R)-4-Amino-5-biphenyl-4-yl-2-hydroxypentanoic acid ethyl ester (HClsalt; 60.3 mg, 0.2 mmol) and methyl 5-chloro-5-oxovalerate (31.7 mg, 0.2mmol) were combined with DIPEA (84 μL, 0.5 mmol) in DMF (5 mL). Themixture was stirred at room temperature until the reaction was complete(1 hour). The solvent was removed and the residue was dissolved in MeOH(3 mL) and 10N NaOH (250 μL), and stirred at 60° C. until the reactionwas complete (1 hour). Glacial acetic acid (250 μL) was added and theproduct was evaporated under reduced pressure and purified bypreparative HPLC to yield Comparative Compound E (8.7 mg, 100% purity).MS m/z [M+H]⁺ calc'd for C₂₂H₂₅NO₆, 400.17; found 400.2.

The compound of Example 1 and Comparative Compounds C, D, and E weretested as described in Assay 1 and found to have pK_(i) values at humanNEP as follows:

Compound R pK_(i) Example 1 —C(O)—COOH 7.9 Comparative Compound C—C(O)—CH₂—COOH 6.7 Comparative Compound D —C(O)—(CH₂)₂—COOH 7.4Comparative Compound E —C(O)—(CH₂)₃—COOH 7.3The data shows that the compound of Example 1 had higher potency at NEPthan Comparative Compounds C, D, and E.

Comparative Example 3

(2S,4S)-5-Biphenyl-4-yl-4-(2-carboxy-acetylamino)-2-hydroxymethylpentanoicAcid (Comparative Compound F; R=—C(O)—CH₂—COOH)

(2S,4S)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-pentanoic acid (HClsalt; (5 mg, 10 μmol) was dissolved in 1 M aqueous NaOH (119 μL, 119μmol) and slowly added to a solution of methyl malonyl chloride (1.9 μL,18 μmol) and MeCN (0.5 mL, 10 mmol). The resulting solution was stirredat room temperature until the reaction was complete (overnight) and theproduct was purified by preparative HPLC to yield Comparative Compound F(1.0 mg, 95% purity). MS m/z [M+H]⁺ calc'd for C₂₁H₂₃NO₆, 386.15; found386.1.

(2S,4S)-5-Biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-hydroxymethylpentanoicAcid (Comparative Compound G; R=—C(O)—(CH₂)₂—COOH)

(2S,4S)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-pentanoic acid (HClsalt; (5 mg, 10 μmol) was dissolved in 1 M aqueous NaOH (119 μL, 119μmol) and slowly added to a solution of 3-(carbomethoxy)propionylchloride (2.2 μL, 18 μmol) and MeCN (0.5 mL, 10 mmol). The resultingsolution was stirred at room temperature until the reaction was complete(overnight) and the product was purified by preparative HPLC to yieldComparative Compound G (3.4 mg, 95% purity). MS m/z [M+H]⁺ calc'd forC₂₂H₂₅NO₆, 400.17; found 400.3.

(2S,4S)-5-Biphenyl-4-yl-4-(4-carboxy-butyrylamino)-2-hydroxymethylpentanoicAcid (Comparative Compound H; R=—C(O)—(CH₂)₃—COOH)

(2S,4S)-4-Amino-5-biphenyl-4-yl-2-hydroxymethyl-pentanoic acid (HClsalt; (5 mg, 10 μmol) was dissolved in 1 M aqueous NaOH (119 μL, 119μmol) and slowly added to a solution of methyl 5-chloro-5-oxovalerate(2.5 μL, 18 μmol) and MeCN (0.5 mL, 10 mmol). The resulting solution wasstirred at room temperature until the reaction was complete (overnight)and the product was purified by preparative HPLC to yield ComparativeCompound H (3.0 mg, 95% purity). MS m/z [M+H]⁺ calc'd for C₂₃H₂₇NO₆,414.18; found 414.7.

The compound of Example 5A and Comparative Compounds F, G, and H weretested as described in Assay 1 and found to have pK_(i) values at humanNEP as follows:

Compound R pK_(i) Example 5A —C(O)—COOH 9.2 Comparative Compound F—C(O)—CH₂—COOH 8.2 Comparative Compound G —C(O)—(CH₂)₂—COOH 9Comparative Compound H —C(O)—(CH₂)₃—COOH 8.6The data shows that the compound of Example 5A had higher potency at NEPthan Comparative Compounds F, G, and H.

Comparative Example 4

(2R,4R)-4-(2-Carboxy-acetylamino)-5-(3′-chloro-biphenyl-4-yl)-2-hydroxy-pentanoicAcid (Comparative Compound R=—C(O)—CH₂—COOH)

Methyl malonyl chloride (18.5 μL, 172 μmol) was added to a solution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (50.0 mg, 144 μmol) and DIPEA (75.1 μL, 431 μmol) in DCM (1.5 mL,23.4 mmol) and the resulting mixture was stirred at room temperature for30 minutes. The mixture was then concentrated to yield a yellow liquid.1 M Aqueous LiOH (719 μL, 719 μmol) was added dropwise to the oil, andthe mixture was stirred at 60° C. for 1 hour. The mixture wasconcentrated in vacuo and the resulting residue was dissolved in AcOH(1.0 mL) purified by preparative HPLC to yield Comparative Compound I(2.0 mg, 100% purity). MS m/z [M+H]⁺ calc'd for C₂₀H₂₀ClNO₆, 406.10;found 406.1.

(2R,4R)-4-(3-Carboxy-propionylamino)-5-(3′-chloro-biphenyl-4-yl)-2-hydroxy-pentanoicAcid (Comparative Compound J; R=—C(O)—(CH₂)₂—COOH)

3-(Carbomethoxy)propionyl chloride (21.2 μL, 172 μmol) was added to asolution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (50.0 mg, 144 μmol) and DIPEA (75.1 μL, 431 μmol) in DCM (1.5 mL,23.4 mmol) and the resulting mixture was stirred at room temperature for30 minutes. The mixture was then concentrated to yield a yellow liquid.1 M Aqueous LiOH (719 μL, 719 μmol) was added dropwise to the oil, andthe mixture was stirred at 60° C. for 1 hour. The mixture wasconcentrated in vacuo and the resulting residue was dissolved in AcOH(1.0 mL) purified by preparative HPLC to yield Comparative Compound J(31.1 mg, 100% purity). MS m/z [M+H]⁺ calc'd for C₂₁H₂₂ClNO₆, 420.11;found 420.2.

(2R,4R)-4-(4-Carboxy-butyrylamino)-5-(3′-chloro-biphenyl-4-yl)-2-hydroxy-pentanoicAcid (Comparative Compound K; R=—C(O)—(CH₂)₃—COOH)

Methyl 5-chloro-5-oxovalerate (23.8 μL, 172 μmol) was added to asolution of(2R,4R)-4-amino-5-(3′-chlorobiphenyl-4-yl)-2-hydroxypentanoic acid ethylester (50.0 mg, 144 μmol) and DIPEA (75.1 μL, 431 μmol) in DCM (1.5 mL,23.4 mmol) and the resulting mixture was stirred at room temperature for30 minutes. The mixture was then concentrated to yield a yellow liquid.1 M Aqueous LiOH (719 μL, 719 μmol) was added dropwise to the oil, andthe mixture was stirred at 60° C. for 1 hour. The mixture wasconcentrated in vacuo and the resulting residue was dissolved in AcOH(1.0 mL) purified by preparative HPLC to yield Comparative Compound K(29.2 mg, 100% purity). MS m/z [M+H]⁺ calc'd for C₂₂H₂₄ClNO₆, 434.13;found 434.2.

The compound of Example 2A and Comparative Compounds I, J, and K weretested as described in Assay 1 and found to have pK_(i) values at humanNEP as follows:

Compound R pK_(i) Example 2A —C(O)—COOH 9.7 Comparative Compound I—C(O)—CH₂—COOH 8.4 Comparative Compound J —C(O)—(CH₂)₂—COOH 9.5Comparative Compound K —C(O)—(CH₂)₃—COOH 9.3The data shows that the compound of Example 2A had higher potency at NEPthan Comparative Compounds I, J, and K.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

What is claimed is:
 1. A compound of formula I:

where: R¹ is selected from H, —C₁₋₈alkyl,—C₁₋₆alkylene-OC(O)R¹⁰, and

where R¹⁰ is —C₁₋₆alkyl, —O—C₁₋₆alkyl, or —CH(R¹⁵)—NHC(O)O—C₁₋₆alkyl,R¹⁴ is —C₁₋₆alkyl; R¹⁵ is —CH(CH₃)₂; R² is —OR²¹ or —CH₂OR²¹; and R³ isH or —CH₃; where R²¹ is H, —C(O)—C₁₋₆alkyl, —C(O)—CH(R²²)—NH₂,—C(O)—CH(R²²)—NHC(O)O—C₁₋₆alkyl, or —P(O)(OR²³)₂; R²² is H, —CH₃,—CH(CH₃)₂, phenyl, or benzyl; R²³ is H, —C₁₋₆alkyl, or phenyl; Z isselected from —CH— and —N—; R⁴ is selected from H, —C₁₋₈alkyl,—C₁₋₃alkylene-O—C₁₋₈alkyl, —C₁₋₃alkylene-O—C₆₋₁₀aryl, -[(CH₂)₂O]₁₋₃CH₃,and

where R⁴⁴ is —C₁₋₆alkyl; a is 0 or 1; R⁵ is selected from halo, —CH₃,—CF₃, and —CN; b is 0 or an integer from 1 to 3; each R⁶ isindependently selected from halo, —OH, —CH₃, —OCH₃, —CN, and —CF₃; whereeach alkyl group in R¹ and R⁴ is optionally substituted with 1 to 8fluoro atoms; and where the methylene linker on the biphenyl isoptionally substituted with one or two —C₁₋₆alkyl groups or cyclopropyl;or a pharmaceutically acceptable salt thereof.
 2. The compound of claim1, where R¹ is H.
 3. The compound of claim 1, where R¹ is selected fromH, —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —(CH₂)₃CH₃, —(CH₂)₆CH₃, —CH₂CF₃,—(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃, —CH₂OC(O)CH₃, —CH₂OC(O)CH₂CH₃,—CH₂OC(O)(CH₂)₂CH₃, —CH₂OC(O)OCH₂CH₃,—CH₂OC(O)—CH[CH(CH₃)₂]—NHC(O)O—CH₃, and

where R¹⁴ is —CH₃.
 4. The compound of claim 1, where R² is —OR²¹, R³ isH, and R²¹ is H.
 5. The compound of claim 1, where R² is —OR²¹, R³ is—CH₃, and R²¹ is H.
 6. The compound of claim 1, where R² is —CH₂OR²¹, R³is H, and R²¹ is H.
 7. The compound of claim 1, where R² is —CH₂OR²¹, R³is —CH₃, and R²¹ is H.
 8. The compound of claim 1, where R⁴ is H.
 9. Thecompound of claim 1, where R⁴ is selected from H, —CH₂CH₃, —CH(CH₃)₂,—CH₂CH(CH₃)₂, —(CH₂)₃CH₃, —C(CH₃)₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃,—(CH₂)₃—O—CH₂CH₃, —(CH₂)₂—O-phenyl, —(CH₂)₂OCH₃, and

where R⁴⁴ is —CH₃.
 10. The compound of claim 1, where a is 0, or a is 1and R⁵ is halo.
 11. The compound of claim 1, where b is 0, or b is 1 andR⁶ is halo, or b is 2 and each R⁶ is independently selected from haloand —CH₃.
 12. The compound of claim 1, where: a is 0 and b is 0; or a is0, b is 1, and R⁶ is 2′-fluoro, 3′-fluoro, 3′-chloro, or 4′-flouro; or ais 0, b is 2, and R⁶ is 2′-fluoro, 5′-chloro or 2′-methyl, 5′-chloro or2′,5′-dichloro; or a is 1, R⁵ is 3-chloro, and b is 0; or a is 1, R⁵ is3-chloro, b is 1, and R⁶ is 3′-chloro; or a is 1, R⁵ is 3-chloro, b is2, and R⁶ is 2′-fluoro, 5′-chloro.
 13. The compound of claim 1, where R¹is selected from H, —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —(CH₂)₃CH₃,—(CH₂)₆CH₃, —CH₂CF₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃, —CH₂CF₂CF₃, —CH₂OC(O)CH₃,—CH₂OC(O)CH₂CH₃, —CH₂OC(O)(CH₂)₂CH₃, —CH₂OC(O)OCH₂CH₃,—CH₂OC(O)—CH[CH(CH₃)₂]—NHC(O)O—CH₃, and

where R¹⁴ is —CH₃; R⁴ is selected from H, —CH₂CH₃, —CH(CH₃)₂,—CH₂CH(CH₃)₂, —(CH₂)₃CH₃, —C(CH₃)₃, —(CH₂)₂CF₃, —CH₂CF₂CH₃,—(CH₂)₃—O—CH₂CH₃, —(CH₂)₂—O-phenyl, —(CH₂)₂OCH₃, and

where R⁴⁴ is —CH₃; and a is 0 and b is 0; or a is 0, b is 1, and R⁶ is2′-fluoro, 3′-fluoro, 3′-chloro, or 4′-flouro; or a is 0, b is 2, and R⁶is 2′-fluoro, 5′-chloro or 2′-methyl, 5′-chloro or 2′,5′-dichloro; or ais 1, R⁵ is 3-chloro, and b is 0; or a is 1, R⁵ is 3-chloro, b is 1, andR⁶ is 3′-chloro; or a is 1, R⁵ is 3-chloro, b is 2, and R⁶ is 2′-fluoro,5′-chloro.
 14. The compound of claim 5, where R¹ is H or —C₁₋₈alkyl; Zis —N—; R⁴ is H or —C₁₋₈alkyl; and a and b are
 0. 15. The compound ofclaim 14, where R¹ and R⁴ are H.
 16. The compound of claim 6, where R¹is H or —C₁₋₈alkyl; Z is —CH—; R⁴ is H or —C₁₋₈alkyl; a is 0 or a is 1and R⁵ is halo; b is 0 or b is 1 or 2 and R⁶ is halo; and where themethylene linker on the biphenyl is optionally substituted with two —CH₃groups.
 17. The compound of claim 16, where R¹ is H, —CH₂CH₃, or—(CH₂)₃CH₃; R⁴ is H; a is 0 or a is 1 and R⁵ is 3-chloro; b is 0 or b is1 and R⁶ is 2′-fluoro, 3′-fluoro, 3′-chloro, or 4′-flouro.
 18. Thecompound of claim 7, where R¹ is H or —C₁₋₈alkyl; Z is —CH—; R⁴ is H or—C₁₋₈alkyl; a is 0; and b is 0, or b is 1 and R⁶ is halo.
 19. Thecompound of claim 18, where R¹ is H or —CH₂CH₃; R⁴ is H or —CH₂CH(CH₃)₂;and b is 0, or b is 1 and R⁶ is 2′-fluoro, 3′-fluoro, 3′-chloro, or4′-flouro.
 20. The compound of claim 1, where R² is —OR²¹ or —CH₂OR²¹;and R³ is H or —CH₃; where R²¹ is H.
 21. The compound of claim 1, wherethe compound has the formula:

or a pharmaceutically acceptable salt thereof.
 22. The compound of claim21, where R¹ is H.
 23. The compound of claim 21, where R⁴ is C₁₋₈alkyl.24. The compound of claim 21, where R⁴ is CH₂CH₃.
 25. The compound ofclaim 21, where R²¹ is H.
 26. The compound of claim 21, where Z is —CH—.27. The compound of claim 21, where a is
 0. 28. The compound of claim21, where b is
 2. 29. The compound of claim 21, where a is 0; b is 2;and R⁶ is 2′-fluoro, 5′-chloro.
 30. A pharmaceutical compositioncomprising the compound of claim 1 or claim 21 and a pharmaceuticallyacceptable carrier.
 31. The pharmaceutical composition of claim 30,further comprising a therapeutic agent selected from adenosine receptorantagonists, α-adrenergic receptor antagonists, β₁-adrenergic receptorantagonists, β₂-adrenergic receptor agonists, dual-acting β-adrenergicreceptor antagonist/α₁-receptor antagonists, advanced glycation endproduct breakers, aldosterone antagonists, aldosterone synthaseinhibitors, aminopeptidase N inhibitors, androgens,angiotensin-converting enzyme inhibitors and dual-actingangiotensin-converting enzyme/neprilysin inhibitors,angiotensin-converting enzyme 2 activators and stimulators,angiotensin-II vaccines, anticoagulants, anti-diabetic agents,antidiarrheal agents, anti-glaucoma agents, anti-lipid agents,antinociceptive agents, anti-thrombotic agents, AT₁ receptor antagonistsand dual-acting AT₁ receptor antagonist/neprilysin inhibitors andmultifunctional angiotensin receptor blockers, bradykinin receptorantagonists, calcium channel blockers, chymase inhibitors, digoxin,diuretics, dopamine agonists, endothelin converting enzyme inhibitors,endothelin receptor antagonists, HMG-CoA reductase inhibitors,estrogens, estrogen receptor agonists and/or antagonists, monoaminereuptake inhibitors, muscle relaxants, natriuretic peptides and theiranalogs, natriuretic peptide clearance receptor antagonists, neprilysininhibitors, nitric oxide donors, non-steroidal anti-inflammatory agents,N-methyl d-aspartate receptor antagonists, opioid receptor agonists,phosphodiesterase inhibitors, prostaglandin analogs, prostaglandinreceptor agonists, renin inhibitors, selective serotonin reuptakeinhibitors, sodium channel blocker, soluble guanylate cyclasestimulators and activators, tricyclic antidepressants, vasopressinreceptor antagonists, and combinations thereof.
 32. The pharmaceuticalcomposition of claim 30, further comprising an AT₁ receptor antagonist.33. The pharmaceutical composition of claim 32, wherein the AT₁ receptorantagonist is selected from abitesartan, azilsartan, azilsartanmedoxomil, benzyllosartan, candesartan, candesartan cilexetil,elisartan, embusartan, enoltasosartan, eprosartan, EXP3174, fonsartan,forasartan, glycyllosartan, irbesartan, isoteoline, losartan, medoximil,milfasartan, olmesartan, olmesartan medoxomil, opomisartan, pratosartan,ripisartan, saprisartan, saralasin, sarmesin, TAK-591, tasosartan,telmisartan, valsartan and zolasartan; or a pharmaceutically acceptablesalt thereof.
 34. The pharmaceutical composition of claim 30, furthercomprising a phosphodiesterase (PDE) inhibitor.
 35. The pharmaceuticalcomposition of claim 30, further comprising a renin inhibitor.
 36. Thepharmaceutical composition of claim 30, further comprising a diuretic.37. A method for treating hypertension, heart failure, or renal disease,comprising administering to a patient a therapeutically effective amountof the compound of claim 1 or claim 21.